Plant cell culture

ABSTRACT

Plant cell cultures as well as related methods, systems, and compositions for increasing the frequency and efficiency of plant genome editing are provided. Various plant cell growth conditions and/or treatments where such increases in gene editing frequencies are obtained are disclosed.

FIELD

Aspects of this disclosure relate to agricultural biotechnology.Disclosed herein are methods useful in the culture and genomic editingof plants, plant parts, plant cells, and plant protoplasts.

INCORPORATION OF SEQUENCE LISTING

A sequence listing containing the file named “63200-172185_ST25.txt”which is 9,937 bytes (measured in MS-Windows®) and created on Feb. 27,2018, comprises 17 nucleotide sequences, is provided herewith via theUSPTO's EFS system and is herein incorporated by reference in itsentirety.

BACKGROUND

Recent advances in genome editing technologies have providedopportunities for precise modification of the genome in many types oforganisms, including plants and animals. For example, technologies basedon genome editing proteins, such as zinc finger nucleases, TALENs, andCRISPR systems are advancing rapidly and it is now possible to targetgenetic changes to specific DNA sequences in the genome. Methods forgrowing and manipulating plant cells and plant protoplasts, includingisolated cells and protoplasts, are especially useful for genome editingas well as genetic engineering technologies.

SUMMARY

Disclosed herein are methods of culturing plant cells or plantprotoplasts having improved viability and/or increased gene-editingfrequencies under cell culture conditions, valuable for use in planttissue culture and plant biotechnology. Also disclosed are compositionsderived from such plant cells or plant protoplasts having improvedviability and/or increased gene-editing frequencies, such as novel plantcells or plant protoplasts, plant callus, plant tissues or parts, wholeplants, and seeds having one or more altered genetic sequences. Furtherdisclosed herein are plant cell cultures, methods, and systems thatprovide for increased plant gene-editing frequencies in plant cells,populations of plant cells with increased plant gene-editingfrequencies, as well as for plant cells, plants, and plant parts withgenome modifications.

In one aspect, the disclosure provides a plant cell or a plantprotoplast culture including: (a) at least one plant cell or one plantprotoplast; and (b) a culture medium including (i) a non-conventionallyhigh concentration (such as at least 30, at least 40, at least 60, atleast 80, or at least 100 millimolar) of a divalent cation, or (ii) anantioxidant, such as, but not limited to, a low-molecular-weight thiolantioxidant; or (iii) a combination of (i) and (ii). In an embodiment,the culture includes (a) at least one plant cell or one plantprotoplast; and (b) a culture medium including: (i) at least 40millimolar Ca²⁺ or Mg²⁺; (ii) an antioxidant; or (iii) a combination of(i) and (ii). Specific embodiments include plant protoplast cultureswherein the culture medium contains from between or about 40-100millimolar Ca²⁺ or Mg²⁺ or 1 millimolar glutathione. Certain embodimentsinclude plant cell cultures wherein the culture medium contains from 30,40, 50, or 60 to 80, 100, 120, or 150 millimolar Ca²⁺ and/or Mg²⁺ and/or0.1, 0.2. or 0.4 to 0.6, 0.8, 1, 1.2, 1.4, 1,6, 1.8, or millimolar of alow-molecular-weight anti-oxidant or low-molecular-weight thiol. Theplant cell or plant protoplast is obtained from any plant part or tissueor callus, and from any monocot or dicot plant species of interest, forexample, row crop plants, fruit-producing plants and trees, vegetables,trees, and ornamental plants including ornamental flowers, shrubs,trees, groundcovers, and turf grasses. In certain embodiments, theculture includes plant cells or plant protoplasts that are encapsulatedor enclosed in a polymer or in a vesicle or liposome or other fluidcompartment; in other embodiments the plant cells or plant protoplastsare not encapsulated. In many embodiments, the culture is in a liquidmedium; in other embodiments, the culture is in a solid or semi-solidmedium, or in a combination of liquid and solid or semi-solid media. Incertain embodiments, the viability of the protoplasts in the culture isimproved, e. g., by at least 10% after at least about one day of culturetime, when compared to the viability of protoplasts in control cultureswithout (i) a relatively high concentration (such as at least 20millimolar) of a divalent cation, or (ii) an antioxidant, such as, butnot limited to, a low-molecular-weight thiol antioxidant; or (iii) acombination of (i) and (ii). In certain embodiments, the culture mediumis maintained under hypoxic conditions, e. g., under about one-halfnormal atmospheric oxygen concentrations or less, for example, atbetween or about 5 to about 10%, or about 5 to about 10%, oxygen byvolume. In certain embodiments, the cell division rate of theprotoplasts is improved compared to that of protoplasts in controlcultures without (i) a relatively high concentration (such as at least20 millimolar) of a divalent cation, or (ii) an antioxidant, such as,but not limited to, a low-molecular-weight thiol antioxidant; or (iii)hypoxic conditions; or (iv) any combination of (i), (ii), and (iii).

In another embodiment, the disclosure provides a method of improvingviability of a plant protoplast, including the step of including in theculture conditions of the protoplast (i) a relatively high concentration(such as at least 20 millimolar) of a divalent cation, or (ii) anantioxidant, such as, but not limited to, a low-molecular-weight thiolantioxidant; or (iii) a combination of (i) and (ii). In certainembodiments of the method, viability of a plant protoplast is improvedby including in the protoplast culture medium at least one of: (a) atleast 40 millimolar Ca²⁺ or Mg²⁺; and (b) at least 1 millimolarlow-molecular-weight thiol. In certain embodiments of the method,viability of a plant protoplast is improved by including in theprotoplast culture medium between or about 40-100 millimolar Ca²⁺ orMg²⁺ or about 1 millimolar glutathione. In certain embodiments of themethod, viability of a plant protoplast is improved, e. g., by at least10% after at least about one day of culture time, when compared to theviability of protoplasts in control cultures without (i) a relativelyhigh concentration (such as at least 20 millimolar) of a divalentcation, or (ii) an antioxidant, such as, but not limited to, alow-molecular-weight thiol antioxidant; or (iii) a combination of (i)and (ii). In certain embodiments of the method, the culture conditionsfurther include use of hypoxic conditions, e. g., wherein the plantprotoplasts are cultured under about one-half normal atmospheric oxygenconcentrations or less, for example, at between or about 5 to about 10%oxygen by volume. In certain embodiments, the cell division rate of theprotoplasts is improved compared to that of protoplasts in controlcultures without (i) a relatively high concentration (such as at least20 millimolar) of a divalent cation, or (ii) an antioxidant, such as,but not limited to, a low-molecular-weight thiol antioxidant; or (iii)hypoxic conditions; or (iv) any combination of (i), (ii), and (iii).Related aspects of the disclosure include the protoplast having improvedviability, increased gene-editing frequencies, and/or improved celldivision rates, as provided by these methods, as well as living plantmaterial (e. g., callus, a somatic embryo, plantlets, plants, seeds, orprogeny plants of future generations) grown or regenerated from such aprotoplast.

In another embodiment, the disclosure provides a method of improving thecell division rate of a plant cell or plant protoplast culture, whereinthe culture conditions comprise hypoxic conditions, i. e., where thecells or protoplasts are grown at less than normal atmospheric oxygenconditions (less than about 21% oxygen by volume). In certainembodiments, plant cells or plant protoplasts are cultured at aboutone-half normal atmospheric oxygen conditions, or at about 10% oxygen byvolume, or at about 5% oxygen by volume, or at between or about 5% toabout 10% oxygen by volume, or between or about 1% to about 5% oxygen byvolume, or between or about 1% to about 10% oxygen by volume. In certainembodiments, the culture conditions further comprise including in theculture medium a relatively high level of divalent cations, for example,at least 10 millimolar divalent cations (e. g., at least 40 millimolarCa²⁺ or Mg²⁺). In certain embodiments, the culture conditions furthercomprise including in the culture medium an antioxidant, such as anantioxidant thiol (e. g., between or about at least about 0.1 to about 1millimolar antioxidant thiol, or between or about at least about 0.5 toabout 100 millimolar antioxidant thiol such as glutathione). In certainembodiments, the cell division rate of the plant cell or plantprotoplast culture subjected to or grown under hypoxic conditions isimproved by at least 10%, at least 15%, at least 25%, at least 50%, atleast 75%, at least 100%, or by at least 2-fold, in comparison to asimilar culture subjected to or grown under non-hypoxic conditions.

Protoplasts having improved viability, increased gene editingfrequencies, and/or improved cell division rates as provided by themethods and culture conditions described herein are useful in planttissue culture and plant biotechnology, e.g., in methods involvinggenetic engineering or genome editing. Thus, in another aspect, thedisclosure provides a composition or plant cell culture including: (a)at least one plant cell or protoplast having improved viability and/orincreased gene-editing frequencies, provided by including in the cultureconditions of the protoplast (i) a relatively high concentration (suchas at least 20 millimolar) of a divalent cation, or (ii) an antioxidant,such as, but not limited to, a low-molecular-weight thiol antioxidant;or (iii) hypoxic conditions; or (iv) any combination of (i), (ii), and(iii); (b) at least one effector molecule (e. g., a polynucleotide or aprotein or a combination of both) for inducing a genetic alteration inthe plant cell or plant protoplast; and (c) optionally, at least onedelivery agent (such as at least one chemical, enzymatic, or physicalagent). Certain embodiments include plant cell cultures or compositionscomprising; (a) at least one plant cell or protoplast having improvedviability and/or increased gene-editing frequencies provided byincluding in the culture conditions of the protoplast (i) a relativelyhigh concentration (such as at least 20 millimolar) of a divalentcation, or (ii) an antioxidant, such as, but not limited to, alow-molecular-weight thiol antioxidant; or (iii) hypoxic conditions; or(iv) any combination of (i), (ii), and (iii); (b) gene editing molecules(e.g., (i) an RNA-guided nuclease and a guide RNA and optionally a donortemplate polynucleotide; (ii) a sequence-specific endonuclease and adonor template polynucleotide; (iii) one or more polynucleotidesencoding an RNA-guided nuclease and a guide RNA; (iv) one or morepolynucleotide(s) encoding a sequence-specific endonuclease and a donortemplate polynucleotide; or (v) any combination thereof); and (c)optionally, at least one delivery agent (such as at least one chemical,enzymatic, or physical agent). Embodiments include compositionsincluding at least one protoplast having improved viability and/orincreased gene-editing frequencies and an RNA guide for an RNA-guidednuclease (or a polynucleotide encoding an RNA guide for an RNA-guidednuclease) and/or an RNA-guided DNA nuclease (or a polynucleotideencoding an RNA-guided DNA nuclease); optionally such compositionsfurther include at least one chemical, enzymatic, or physical deliveryagent. In a related aspect, the disclosure provides arrangements ofprotoplasts having improved viability and/or improved cell divisionrates as provided by the methods and culture conditions describedherein, such as arrangements of protoplasts convenient for screeningpurposes. In an aspect, the disclosure provides an array including aplurality of containers, each including at least one protoplast havingimproved viability, increased gene-editing frequencies, and/or improvedcell division rates, provided by including in the culture conditions ofthe protoplast (i) a relatively high concentration (such as at least 20millimolar) of a divalent cation, or (ii) an antioxidant, such as, butnot limited to, a low-molecular-weight thiol antioxidant; or (iii)hypoxic conditions; or (iv) any combination of (i), (ii), and (iii). Incertain embodiments, the disclosure provides compositions comprising:(a) at least one plant cell or plant protoplast having improvedviability and/or increased gene-editing frequencies; (b) at least oneeffector molecule for inducing a genetic alteration in the plant cell orplant protoplast, wherein the at least one effector molecule is selectedfrom the group consisting of: (i) a polynucleotide selected from thegroup consisting of an RNA guide for an RNA-guided nuclease, a DNAencoding an RNA guide for an RNA-guided nuclease; (ii) a nucleaseselected from the group consisting of an RNA-guided nuclease, anRNA-guided DNA endonuclease, a type II Cas nuclease, a Cas9, a type VCas nuclease, a Cpfl, a CasY, a CasX, a C2c1, a C2c3, an engineerednuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN), atranscription activator-like effector nuclease (TAL-effector nuclease),Argonaute, a meganuclease or engineered meganuclease; (iii) apolynucleotide encoding one or more of any aforementioned or othernuclease capable of effecting site-specific alteration of a targetnucleotide sequence; and/or (iv) at least one donor templatepolynucleotide; (c) at least one of: (i) at least 40 millimolar Ca²⁺ orMg²⁺; and/or (ii) at least 1 millimolar low-molecular-weight thiol, and;(e) optionally, at least one delivery agent selected from the groupconsisting of solvents, fluorocarbons, glycols or polyols, surfactants;primary, secondary, or tertiary amines and quaternary ammonium salts;organosilicone surfactants; lipids, lipoproteins, lipopolysaccharides;acids, bases, caustic agents; peptides, proteins, or enzymes;cell-penetrating peptides; RNase inhibitors; cationic branched or linearpolymers; dendrimers; counter-ions, amines or polyamines, osmolytes,buffers, and salts; polynucleotides; transfection agents; antibiotics;non-specific DNA double-strand-break-inducing agents; and antioxidants;particles or nanoparticles, magnetic particles or nanoparticles,abrasive or scarifying agents, needles or microneedles, matrices, grids,and combinations thereof. In certain embodiments of any of theaforementioned compositions or plant cell cultures, the genome editingmolecule(s) comprise: (i) an RNA-guided nuclease and a guide RNA andoptionally a donor template polynucleotide; (ii) a sequence-specificendonuclease and a donor template polynucleotide; (iii) one or morepolynucleotides encoding an RNA-guided nuclease and a guide RNA; (iv)one or more polynucleotide(s) encoding a sequence-specific endonucleaseand a donor template polynucleotide; or (v) any combination thereof.

Methods are provided for making a plant cell having a genomicmodification comprising: (a) providing genome editing molecules to aplant cell previously, concurrently, or subsequently exposed to ahypoxic condition, a reactive oxygen species (ROS) concentrationlowering agent, or combination thereof; wherein the molecules comprise:(i) an RNA-guided nuclease and a guide RNA and optionally a donortemplate polynucleotide; (ii) a sequence-specific endonuclease and adonor template polynucleotide; (iii) one or more polynucleotidesencoding an RNA-guided nuclease and a guide RNA and optionally a donortemplate polynucleotide; (iv) a polynucleotide encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof, to modify the plant cell's genome; and, (b)isolating or propagating a plant cell comprising the genomemodification. In certain embodiments, the methods further compriseobtaining callus, a propagule, or a plant from the isolated orpropagated plant cell of step (b) comprising the genome modification,wherein the callus, propagule, or plant comprises a genome modified bythe molecule(s) and wherein the propagule is optionally a seed.

Methods are provided for producing a plant having a genomic modificationcomprising: (a) providing genome editing molecules to a plant cellpreviously, concurrently, or subsequently exposed to a hypoxiccondition, a reactive oxygen species (ROS) concentration lowering agent,or combination thereof, wherein the molecules comprise: (i) anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (ii) a sequence-specific endonuclease and a donortemplate polynucleotide; (iii) one or more polynucleotides encoding anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (iv) a polynucleotide encoding a sequence-specificendonuclease and a donor template polynucleotide; or (v) any combinationthereof, to modify the plant cell's genome; (b) isolating or propagatinga plant cell comprising the genome modification; and, (c) regeneratingor obtaining a plant comprising the genome modification from the plantcell. In certain embodiments, the methods further comprise harvestingseed from the plant, propagating the plant, or multiplying the plant.

Systems are provided for producing a plant cell having a genomicmodification comprising: (a) a plant cell subjected to a hypoxiccondition, or treated with a reactive oxygen species (ROS) scavengingagent, or both subjected to the hypoxic condition and treated with theROS scavenging agent; and (b) genome editing molecule(s) comprising: (i)an RNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (ii) a sequence-specific endonuclease and a donortemplate polynucleotide; (iii) one or more polynucleotides encoding anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (iv) one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof; wherein said plant cell is associated with,contacts, and/or contains said molecule(s).

Systems are provided for producing a plant cell having a genomicmodification comprising: (a) a plant cell wherein a reactive oxygenspecies (ROS) concentration is lowered in comparison to a control plantcell; and (b) genome editing molecule(s) comprising: (i) an RNA-guidednuclease and a guide RNA and optionally a donor template polynucleotide;(ii) a sequence-specific endonuclease and a donor templatepolynucleotide; (iii) one or more polynucleotides encoding an RNA-guidednuclease and a guide RNA and optionally a donor template polynucleotide;(iv) one or more polynucleotide(s) encoding a sequence-specificendonuclease and a donor template polynucleotide; or (v) any combinationthereof; wherein said plant cell is associated with, contacts, and/orcontains said molecule(s).

Compositions are provided that comprise: (a) a plant cell wherein areactive oxygen species (ROS) concentration is lowered in comparison toa control plant cell; and (b) genome editing molecule(s) comprising: (i)an RNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (ii) a sequence-specific endonuclease and a donortemplate polynucleotide; (iii) one or more polynucleotides encoding anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (iv) one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof; wherein said plant cell is associated with,contacts, and/or contains said agent and said molecule(s).

Plant cell cultures are provided that comprise: (a) a plant cell culturemedium; (b) a plant cell exposed to a hypoxic condition, or to areactive oxygen species (ROS) concentration lowering agent, or to acombination thereof, wherein the plant cell is contained or supported bythe plant cell culture medium; and, (c) genome editing molecule(s)comprising: (i) an RNA-guided nuclease and a guide RNA and optionally adonor template polynucleotide; (ii) a sequence-specific endonuclease anda donor template polynucleotide; (iii) one or more polynucleotidesencoding an RNA-guided nuclease and a guide RNA and optionally a donortemplate polynucleotide; (iv) one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof; wherein the plant cell is associated with,contacts, and/or contains the molecule(s).

Methods are provided for making a plant cell having a genomicmodification that comprise: (a) providing genome editing molecules to aplant cell previously, concurrently, or subsequently subjected to ahypoxic condition, or to a reactive oxygen species (ROS) concentrationlowering agent, or to a combination thereof; wherein the moleculescomprise: (i) an RNA-guided nuclease and a guide RNA and optionally adonor template polynucleotide; (ii) a sequence-specific endonuclease anda donor template polynucleotide; (iii) one or more polynucleotidesencoding an RNA-guided nuclease and a guide RNA and optionally a donortemplate polynucleotide; (iv) a polynucleotide encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof, to modify the plant cell's genome; and, (b)isolating, selecting, identifying, and/or propagating a plant cellcomprising the genome modification, thereby making the plant cell havinga genomic modification. In certain embodiments, the methods can furthercomprise obtaining callus, a propagule, or a plant from the isolated,selected, identified, and/or propagated plant cell of step (b)comprising the genome modification, wherein the callus, propagule, orplant comprises a genome modified by the molecule(s) and wherein thepropagule is optionally a seed.

Systems are provided for producing a plant cell having a genomicmodification comprising: (a) a plant cell subjected to a hypoxiccondition, or treated with a reactive oxygen species (ROS) scavengingagent, or both subjected to the hypoxic condition and treated with theROS scavenging agent; and (b) genome editing molecule(s) comprising: (i)an RNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (ii) a sequence-specific endonuclease and a donortemplate polynucleotide; (iii) one or more polynucleotides encoding anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (iv) one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof; wherein the plant cell is associated with,contacts, and/or contains the molecule(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts results of an experiment measuring cell division rates inmaize B104 cells as described in detail in Example 12. Cell divisionrates are expressed as “% EdU” (percentage of cells displaying the EdUsignal relative to the total cell count). Dark solid bars representcells grown in medium including 100 millimolar calcium, no addedglutathione (“Glut”), and under hypoxic conditions (5% oxygen byvolume). White solid bars represent cells grown in medium including 100millimolar calcium, no added glutathione (“Glut”), and under normalatmospheric oxygen conditions (21% oxygen by volume). Bars shaded withdiagonal lines represent cells grown in medium including 100 millimolarcalcium, 0.5 millimolar glutathione (“Glut”), and under hypoxicconditions (5% oxygen by volume). Bars shaded with stippling representcells grown in medium including 100 millimolar calcium, 0.5 millimolarglutathione (“Glut”), and under normal atmospheric oxygen conditions(21% oxygen by volume).

DETAILED DESCRIPTION

Unless otherwise stated, nucleic acid sequences in the text of thisspecification are given, when read from left to right, in the 5′ to 3′direction. Nucleic acid sequences may be provided as DNA or as RNA, asspecified; disclosure of one necessarily defines the other, as well asnecessarily defines the exact complements, as is known to one ofordinary skill in the art. Where a term is provided in the singular, theinventors also contemplate embodiments and aspects of the disclosuredescribed by the plural of that term.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the phrase “gene-editing” includes genome modificationby homology directed repair (HDR) mechanisms. Such gene-editing includesembodiments where a site specific nuclease and a donor template areprovided.

As used herein, the terms “expose” or “exposed” and the like aresynonymous with the terms “subject” or “subjected” and the like.

As used herein, an “exogenous” agent or molecule refers to any agent ormolecule from an external source that is provided to or introduced intoa system, composition, plant cell culture, reaction system, or plantcell. In certain embodiments, the exogenous agent (e.g., polynucleotide,protein, or compound) from the external source can be an agent that isalso found in a plant cell. In certain embodiments, the exogenous agent(e.g., polynucleotide, protein, or compound) from the external sourcecan be an agent that is heterologous to the plant cell.

As used herein, a “heterologous” agent or molecule refers to any agentor molecule that is not found in a wild-type, untreated, or naturallyoccurring composition or plant cell; and/or (ii) to a polynucleotide orpeptide sequence located in, e. g., a genome or a vector, in a contextother than that in which the sequence occurs in nature. For example, apromoter that is operably linked to a gene other than the gene that thepromoter is operably linked to in nature is a heterologous promoter.

As used herein, the terms “include,” “includes,” and “including” are tobe construed as at least having the features to which they refer whilenot excluding any additional unspecified features.

As used herein, the phrases “low-molecular-weight antioxidant” or“low-molecule-weight thiol” respectively refer to antioxidant or thiolcompounds having a molecular weight of less than about 1000.

As used herein, the phrase “plant cell” includes plant cells locatedwithin a plant, plant tissue, callus tissue, embryogenic callus, orplant part in an undissociated form, plant cells in a dissociated formor semi-dissociated form that have a cell wall or a portion of a cellwall, as well as plant protoplasts that lack a cell well in adissociated or semi-dissociated form.

As used herein, the phrase “plant cell culture” refers to plant tissue,callus tissue, embryogenic callus, or plant part in an undissociatedform, plant cells in a dissociated form or semi-dissociated form thathave a cell wall or a portion of a cell wall, as well as plantprotoplasts in a dissociated or semi-dissociated form that lack a cellwell, wherein the plant tissue, callus tissue, embryogenic callus, plantpart, plant cells or plant protoplasts are contained in or supported bya plant cell culture medium.

As used herein, the phrase “plant cell culture medium” refers to any ofa liquid, solid, and/or gel that contains nutrients sufficient tosupport viability and/or growth of a plant cell maintained therein orthereon. Examples of plant cell culture medium include compositionscomprising at least salts and vitamins that support plant viabilityand/or growth (e.g., Gamborg's B-5 Basal Medium, Murashige and SkoogBasal Medium (MS), Schenk and Hildebrandt Basal Salt Mixture, and thelike).

By “polynucleotide” is meant a nucleic acid molecule containing 2 ormore nucleotides. Polynucleotides are generally described as single- ordouble-stranded. Where a polynucleotide contains double-stranded regionsformed by intra- or intermolecular hybridization, the length of eachdouble-stranded region is conveniently described in terms of the numberof base pairs. Methods of using polynucleotides or compositionscontaining polynucleotides are provided herein. Embodiments of themethods and compositions provided herein can employ or include: one ormore polynucleotides of 2 to 25 residues in length, one or morepolynucleotides of more than 26 residues in length, or a mixture ofboth. Polynucleotides can comprise single- or double-stranded RNA,single- or double-stranded DNA, double-stranded DNA/RNA hybrids,chemically modified analogues thereof, or a mixture thereof. In certainembodiments, a polynucleotide includes a combination of ribonucleotidesand deoxyribonucleotides (e. g., synthetic polynucleotides consistingmainly of ribonucleotides but with one or more terminaldeoxyribonucleotides or synthetic polynucleotides consisting mainly ofdeoxyribonucleotides but with one or more terminaldideoxyribonucleotides), or includes non-canonical nucleotides such asinosine, thiouridine, or pseudouridine. In certain embodiments, thepolynucleotide includes chemically modified nucleotides (see, e. g.,Verma and Eckstein (1998) Annu. Rev. Biochem., 67:99-134). Chemicallymodified nucleotides that can be used in the polynucleotides providedherein include: (i) phosphorothioate, phosphorodithioate, ormethylphosphonate internucleotide linkage modifications; (ii) modifiednucleoside bases and/or modified sugars; (iii) detectable labelsincluding a fluorescent moiety (e. g., fluorescein or rhodamine or afluorescence resonance energy transfer or FRET pair of chromophorelabels) or other label (e. g., biotin or an isotope). Polynucleotidesprovided or used herein also include modified nucleic acids,particularly modified RNAs, which are disclosed in U.S. Pat. No.9,464,124, incorporated by reference in its entirety herein.

As used herein the term “synergistic” refers to an effect of combiningat least two factors that exceeds the sum of the effects obtained whenthe factors are not combined.

As used herein, the phrase “target plant gene” refers to a gene locatedin the plant genome that is to be modified by gene editing moleculesprovided in a system, method, composition and/or plant cell providedherein. Embodiments of target plant genes include (protein-) codingsequence, non-coding sequence, and combinations of coding and non-codingsequences. Modifications of a target plant gene include nucleotidesubstitutions, insertions, and/or deletions in one or more elements of aplant gene that include a transcriptional enhancer or promoter, a 5′ or3′ untranslated region, a mature or precursor RNA coding sequence, anexon, an intron, a splice donor and/or acceptor, a protein codingsequence, a polyadenylation site, and/or a transcriptional terminator.In certain embodiments, all copies or all alleles of a given target genein a diploid or polyploid plant cell are modified to providehomozygosity of the modified target gene in the plant cell. In certainembodiments, where a desired trait is conferred by a loss-of-functionmutation that is introduced into the target gene by gene editing, aplant cell, population of plant cells, plant, or seed is homozygous fora modified target gene with the loss-of-function mutation. In otherembodiments, only a subset of the copies or alleles of a given targetgene are modified to provide heterozygosity of the modified target genein the plant cell. In certain embodiments where a desired trait isconferred by a dominant mutation that is introduced into the target geneby gene editing, a plant cell, population of plant cells, plant, or seedis heterozygous for a modified target gene with the dominant mutation.Traits imparted by such modifications to certain plant target genesinclude improved yield, resistance to insects, fungi, bacterialpathogens, and/or nematodes, herbicide tolerance, abiotic stresstolerance (e.g., drought, cold, salt, and/or heat tolerance), proteinquantity and/or quality, starch quantity and/or quality, lipid quantityand/or quality, secondary metabolite quantity and/or quality, and thelike, all in comparison to a control plant that lacks the modification.

To the extent to which any of the preceding definitions is inconsistentwith definitions provided in any patent or non-patent referenceincorporated herein by reference, any patent or non-patent referencecited herein, or in any patent or non-patent reference found elsewhere,it is understood that the preceding definition will be used herein.

Systems, methods, and compositions that provide for increasedfrequencies of plant gene editing in comparison to controls are providedherein. Such systems, methods and compositions can comprise acombination at least two features that provide for such increased plantgene editing frequencies. In certain embodiments, a first featurecomprises plant cells that have been exposed to hypoxic conditionsand/or agents that reduce reactive oxygen species (ROS) or plant cellsthat have lowered ROS concentrations. In certain embodiments, a secondfeature comprises treatment of the plant cells that have been exposed tohypoxic conditions and/or agents that reduce ROS or the plant cells thathave lowered ROS concentrations with a non-conventionally highconcentration of a divalent cation or divalent cation mixture.

In one aspect, the disclosure provides a plant cell or a plantprotoplast culture including: (a) at least one plant cell or one plantprotoplast; and (b) a culture medium including (i) a non-conventionallyhigh concentration (such as at least 30, at least 40, at least 60, atleast 80, or at least 100 millimolar) of a divalent cation or divalentcation mixture, or (ii) an antioxidant; or (iii) a combination of (i)and (ii). In an embodiment, the plant cell or a plant protoplast cultureincludes (a) at least one plant cell or one plant protoplast; and (b) aculture medium including: (i) at least 40 millimolar Ca²⁺ or Mg²⁺; (ii)about 0.1 to about 10 millimolar of a low-molecular-weight(non-enzymatic) antioxidant, such as, but not limited to, alow-molecular-weight thiol antioxidant; or (iii) a combination of (i)and (ii). Embodiments include plant cell or plant protoplast cultureswherein the culture medium contains:

-   -   (a) between about 40 to about 60 millimolar, or about 40 to        about 60 millimolar, Ca²⁺ and/or Mg²⁺;    -   (b) between about 40 to about 80 millimolar, or about 40 to        about 80 millimolar, Ca²⁺ and/or Mg²⁺;    -   (c) between about 40 to about 100 millimolar, or about 40 to        about 100 millimolar, Ca²⁺ and/or Mg²⁺;    -   (d) between about 40 to about 150 millimolar, or about 40 to        about 150 millimolar, Ca²⁺ and/or Mg²⁺;    -   (e) between about 60 to about 80 millimolar, or about 60 to        about 80 millimolar, Ca²⁺ and/or Mg²⁺;    -   (f) between about 60 to about 100 millimolar, or about 60 to        about 100 millimolar, Ca²⁺ and/or Mg²⁺;    -   (g) between about 60 to about 150 millimolar, or about 60 to        about 150 millimolar, Ca²⁺ and/or Mg²⁺;    -   (h) between about 80 to about 100 millimolar, or about 80 to        about 100 millimolar, Ca²⁺ and/or Mg²⁺;    -   (i) between about 80 to about 150 millimolar, or about 80 to        about 150 millimolar, Ca²⁺ and/or Mg²⁺;    -   (j) between about 0.1 to about 1 millimolar, or about 0.1 to        about 1 millimolar, low-molecular-weight antioxidant;    -   (k) between about 1 to about 10 millimolar, or about 1 to about        10 millimolar, low-molecular-weight antioxidant;    -   (l) between about 0.1 to about 1 millimolar, or about 0.1 to        about 1 millimolar, low-molecular-weight thiol (e. g.,        glutathione, cysteine, cysteinyl glycine, gamma-glutamyl        cysteine, N-acetylcysteine, cysteine, thiocysteine,        homocysteine, lipoic acid, and dithiothreitol); and/or    -   (m) between about 1 to about 10 millimolar, or about 1 to about        10 millimolar, low-molecular-weight thiol (e. g., glutathione,        cysteine, cysteinyl glycine, gamma-glutamyl cysteine,        N-acetylcysteine, cysteine, thiocysteine, homocysteine, lipoic        acid, and dithiothreitol).

Embodiments of the methods, systems, or compositions provided hereininclude plant cell cultures having any of the aforementioned divalentcation and/or antioxidants set forth in (a), (b), (c), (d), (e), (f),(g), (h), (i), (j), and/or (k).

Embodiments of the systems, methods, or compositions provided hereininclude cultures, plants, and/or plant parts wherein the plant cell isexposed or treated with an enzymatic and/or a non-enzymatic ROSscavenging agent. In certain embodiments, such exposure or treatmentwith the enzymatic and/or a non-enzymatic ROS scavenging agent resultsin lowered concentrations of ROS (e.g., hydrogen peroxide, a superoxideradical, a peroxide ion, a hydroperoxyl radical, and/or a hydroxylradical) in the exposed or treated plant cell in comparison to anunexposed or untreated plant cell. In certain embodiments, thenon-enzymatic ROS scavenging agents include low-molecular-weightantioxidants, including lipid-soluble antioxidants and water-solubleantioxidants (e.g., low-molecular-weight thiol antioxidants, pro-thiols,ascorbic acid, tocopherols, carotenoids, flavonoids, butylatedhydroxytoluene, and butylated hydroxyanisole). In certain embodiments,the non-enzymatic ROS scavenging agents are provided at a concentrationof about 0.1 to about 10 millimolar. Specific embodiments includecultures wherein the culture medium includes about 0.1 to about 10millimolar low-molecular-weight thiol antioxidants. Low-molecular-weightthiol antioxidants useful in the systems, methods, and compositionsinclude glutathione (gamma-glutamylcysteinyl glycine), cysteine,cysteinyl glycine, gamma-glutamyl cysteine, N-acetylcysteine, cysteine,thiocysteine, homocysteine, lipoic acid, and/or dithiothreitol (any ofwhich can also be used in combination with each other at a similar finalthiol concentration). ROS scavenging agents useful in the systems,methods, and compositions also include pro-thiols (e.g.,L-2-oxothiazolidine-4-carboxylate (OTC)) which are converted to thiolsin the cell. In certain embodiments, the plant cell is exposed ortreated with enzymatic ROS scavenging agents. Enzymatic ROS scavengingagents include any catalase, ascorbate peroxidase, a dehydroascorbatereductase, guaiacol peroxidase, monodehydroascorbate reductase, aperoxidase, and/or superoxide dismutase. In certain embodiments, anenzymatic ROS scavenging agents is provided in the culture medium. Incertain embodiments, an enzymatic ROS scavenging agent orpolynucleotides encoding the same can be introduced into the plant cell(e.g., by transient or stable transformation, transfection, or with adelivery agent). A combination of at least one enzymatic and at leastone non-enzymatic ROS scavenging agent can also be used. Specificembodiments also include plant cell or plant protoplast cultures whereinthe culture medium includes about 20, about 40, or about 60 to about 80,about 100, about 120, or about 150 millimolar Ca²⁺ and/or Mg²⁺ and/or inwhich the culture medium includes about 0.1, about 0.25, about 0.5,about 0.75, about 1, or about 2 to about 4, about 6, about 8, or about10 millimolar low-molecular-weight thiol antioxidant. Furtherembodiments encompassed are plant cell or plant protoplast cultureswherein the culture medium includes combinations of divalent cations andlow-molecular-weight antioxidants, with the individual componentspresent in the culture at concentrations similar to those listed above.In certain embodiments, the plant cells (e.g., plant protoplasts) areexposed to the ROS scavenging agents about 5, 10, 15, 30, or 45 minutesto about 60, 75, 90, or 120 minutes after exposure to the gene-editingmolecules. In certain embodiments, the plant cells (e.g., plantprotoplasts) are exposed to the ROS scavenging agents prior to or at thesame time that they are exposed to the gene-editing molecules.

Embodiments of the methods, systems, or compositions provided hereinalso include plant cell or plant protoplast cultures wherein the culturemedium includes at least 40 millimolar Ca²⁺, or at least 50 millimolarCa²⁺, or at least 100 millimolar Ca²⁺. Embodiments include plant cell orplant protoplast cultures wherein the culture medium includes at least40 millimolar Mg²⁺, or at least 50 millimolar Mg²⁺, or at least 100millimolar Mg²⁺. Embodiments include plant cell or plant protoplastcultures wherein the culture medium includes between or about 0.1 toabout 10 millimolar low-molecular-weight antioxidants, includinglipid-soluble antioxidants and water-soluble antioxidants, for example,low-molecular-weight thiol antioxidants, ascorbic acid, tocopherols,butylated hydroxytoluene, and butylated hydroxyanisole. Specificembodiments include plant cell or plant protoplast cultures wherein theculture medium includes between about 0.1 to about 10 millimolarlow-molecular-weight thiol antioxidants see, e. g., Pivato et al. (2014)Archives Biochem. Biophys., 560:83-99. Low-molecular-weight thiolantioxidants useful in compositions and methods provided herein include,but are not limited to, glutathione (gamma-glutamylcysteinyl glycine),cysteine, cysteinyl glycine, gamma-glutamyl cysteine, N-acetylcysteine,cysteine, thiocysteine, homocysteine, lipoic acid, and dithiothreitol(any of which can also be used in combination with each other at asimilar final thiol concentration). Specific embodiments include plantcell or plant protoplast cultures wherein the culture medium includesabout 20, about 40, about 60, about 80, about 100, about 120, or about150 millimolar Ca²⁺, or in which the culture medium includes about 0.1,about 0.25, about 0.5, about 0.75, about 1, about 2, about 4, about 6,about 8, or about 10 millimolar low-molecular-weight thiol antioxidant.Further embodiments encompassed are plant cell or plant protoplastcultures wherein the culture medium includes combinations of divalentcations and low-molecular-weight antioxidants or low-molecular-weightthiols, with the individual components present in the culture atconcentrations similar to those listed above.

In certain embodiments of the methods, systems, and compositionsprovided herein, the culture medium is maintained under hypoxicconditions, e. g., under about one-half normal atmospheric oxygenconcentrations or less, for example, at between or about 5 to about 10%oxygen by volume. Normal (i.e., “normoxic”) oxygen conditions compriseabout 21% oxygen by volume. Hypoxic conditions used in the systems,methods, and compositions provided herein can in certain embodimentscomprise about 14%, 13%, 12%, 11%, or 10% to about 8%, 7%, 6%, or 5%oxygen by volume. In certain embodiments, hypoxic conditions cancomprise treating the plant cells with a hypoxia mimetic (e.g.,desferrioxamine or cobalt chloride). In certain embodiments, a hypoxiccondition can comprises maintaining the cell in a liquid culture mediahaving a dissolved oxygen concentration that is lower than the dissolvedoxygen concentration obtained when the liquid culture media is undernormoxic conditions. Such exposure of the plant cell to the hypoxiccondition can in certain embodiments be limited to a period of timenecessary to realize improvements in gene editing frequencies (e.g.,prior to and/or during association, contact, and/or containment to/of anROS lowering agent and/or gene editing molecule; prior to and/or duringexposure and/or after to an ROS lowering agent and/or gene editingmolecule). Such exposure and or maintenance of a plant cell underhypoxic conditions can be achieved in the context of a plant cell inisolated form (e.g., as a protoplast), a plant cell in a plant embryo,plant callus, especially embryogenic callus, in an isolated plant tissueor part (e.g., an ovule, anther, leaf, meristematic tissue, and thelike), or in a whole plant. In certain embodiments, the plant cell inany of the aforementioned contexts can be in a liquid or solid culturemedium that includes about 20, about 40, or about 60 to about 80, about100, about 120, or about 150 millimolar Ca²⁺ and/or Mg²⁺, and is exposedto and/or maintained under hypoxic conditions. In certain embodiments,the plant cells (e.g., plant protoplasts) are exposed to the hypoxicconditions about 5, 10, 15, 30, or 45 minutes to about 60, 75, 90, or120 minutes after exposure to the gene-editing molecules and/or ROSlowering agent In certain embodiments, the culture medium includes about20, about 40, about 60, about 80, about 100, about 120, or about 150millimolar Ca²⁺ or Mg²⁺, and is maintained under hypoxic conditions. Incertain embodiments, the culture medium includes between or about 40 toabout 150 millimolar Ca²⁺, and is maintained between or about 5 to about10% oxygen by volume. In certain embodiments, the cell division rate ofthe protoplasts is improved compared to that of protoplasts in controlcultures without (i) a relatively high concentration (such as at least20 millimolar) of a divalent cation, or (ii) an antioxidant, such as,but not limited to, a low-molecular-weight thiol antioxidant; or (iii)hypoxic conditions; or (iv) any combination of (i), (ii), and (iii). Incertain embodiments, the cell division rate of the plant cells or plantprotoplasts is improved by at least 20%, or by at least 50%, or by atleast 75%, or by at least 100%. In an embodiment, the culture conditionsinclude at least 40 millimolar Ca²⁺ and about one-half normalatmospheric oxygen concentrations, and the cell division rate of theplant cells or plant protoplasts is improved by at least 100% (i. e.,cell division rate is about twice that observed in similar culturesgrown under or subjected to normal atmospheric oxygen concentrations).

In certain embodiments, the plant cell or plant protoplast cultures areexposed to the aforementioned culture media immediately afterintroduction of a gene editing molecule. In certain embodiments, theplant cell or plant protoplast cultures are exposed to theaforementioned culture media during the time that they are treated witha gene editing molecule and immediately afterwards. In certainembodiments, the plant cell or plant protoplast cultures are exposed tothe aforementioned culture media before and/or during the time that theyare treated with a gene editing molecule and/or immediately afterwards.Exposure of the plant cell or plant protoplast cultures to the culturemedia can be for about 1, 2, 4, 6, or 8 to about 12, 18, 24, 36, or 48hours after introduction of a gene editing molecule. Gene editingmolecules can be introduced by methods that include transfection,Agrobacterium-mediated transformation, Agro-infection, electroporation,and the like. In certain embodiments, the plant cell or plant protoplastis maintained at a temperature of about 30° C., 32° C., 34° C., or 36°C. to about 38° C., 40° C., or 42° C. for at least about 30, 40, 50, or60 minutes, or for about 30, 40, 50, 60, to about 70, 80, 90, or 120minutes, following introduction of the gene editing molecules.

In certain embodiments, plant cells or plant protoplasts in the culturesystem, method, composition or reaction mixtures provided herein aregenerally isolated plant cells or plant protoplasts, that is to say, notlocated in undissociated or intact plant tissues, plant parts, or wholeplants. In certain embodiments, the culture includes plant cells orplant protoplasts obtained from any plant part or tissue or callus. Incertain embodiments, the culture includes plant cells or plantprotoplasts obtained from a plant tissue, whole plant, intact nodal bud,shoot apex or shoot apical meristem, root apex or root apical meristem,lateral meristem, intercalary meristem, seedling, whole seed, halvedseed or other seed fragment, zygotic embryo, somatic embryo, ovule,pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem,tuber, root, callus, or plant cell suspension.

In certain embodiments, plant cells in the system, method, compositionor reaction mixtures provided herein are plant cells that are located inundissociated or intact plant tissues, plant parts, or whole plants. Incertain embodiments, the plant cell can be located in an intact nodalbud, shoot apex or shoot apical meristem, root apex or root apicalmeristem, lateral meristem, intercalary meristem, seedling, whole seed,halved seed or other seed fragment, zygotic embryo, somatic embryo,ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole,stem, tuber, root, or callus.

In embodiments, the culture includes haploid, diploid, or polyploidplant cells or plant protoplasts, for example, those obtained from ahaploid, diploid, or polyploid plant, plant part or tissue, or callus.In certain embodiments, the plant cells or plant protoplasts in theculture (or a regenerated plant, progeny seed, and progeny plantobtained from the plant cells or protoplasts) are haploid or can beinduced to become haploid; techniques for making and using haploidplants and plant cells are known in the art, see, e. g., methods forgenerating haploids in Arabidopsis thaliana by crossing of a wild-typestrain to a haploid-inducing strain that expresses altered forms of thecentromere-specific histone CENH3, as described by Maruthachalam andChan in “How to make haploid Arabidopsis thaliana”, protocol availableatwww[dot]openwetware[dot]org/images/d/d3/Haploid_Arabidopsis_protocol[dot]pdf;Ravi et al. (2014) Nature Communications, 5:5334, doi:10.1038/ncomms6334). Haploids can also be obtained in a wide variety ofmonocot plants (e.g., maize, wheat, rice, sorghum, barley) or dicotplants (e.g., soybean, Brassica sp. including canola, cotton, tomato) bycrossing a plant comprising a mutated CENH3 gene with a wildtype diploidplant to generate haploid progeny as disclosed in U.S. Pat. No.9,215,849, which is incorporated herein by reference in its entirety.Haploid-inducing maize lines that can be used to obtain haploid maizeplants and/or cells include stock 6, MHI (Moldovian Haploid Inducer),indeterminate gametophyte (ig) mutation, KEMS, RWK, ZEM, ZMS, KMS, andwell as transgenic haploid inducer lines disclosed in U.S. Pat. No.9,677,082, which is incorporated herein by reference in its entirety.Examples of haploid cells include but are not limited to plant cellsobtained from haploid plants and plant cells obtained from reproductivetissues, e. g., from flowers, developing flowers or flower buds,ovaries, ovules, megaspores, anthers, pollen, and microspores. Incertain embodiments where the plant cell or plant protoplast is haploid,the genetic complement can be doubled by chromosome doubling (e. g., byspontaneous chromosomal doubling by meiotic non-reduction, or by using achromosome doubling agent such as colchicine, oryzalin, trifluralin,pronamide, nitrous oxide gas, anti-microtubule herbicides,anti-microtubule agents, and mitotic inhibitors) in the plant cell orplant protoplast to produce a doubled haploid plant cell or plantprotoplast wherein the complement of genes or alleles is homozygous; yetother embodiments include regeneration of a doubled haploid plant fromthe doubled haploid plant cell or plant protoplast. Another aspect ofthe disclosure is related to a hybrid plant having at least one parentplant that is a doubled haploid plant provided by this approach.Production of doubled haploid plants provides homozygosity in onegeneration, instead of requiring several generations of self-crossing toobtain homozygous plants; this may be particularly advantageous inslow-growing plants, such as fruit and other trees, or for producinghybrid plants that are offspring of at least one doubled-haploid plant.

In certain embodiments of the methods or systems provided herein, aplant cell or protoplast containing a genomic modification or acomposition or plant cell culture comprising the plant cell orprotoplast containing a genomic modification, can be used to obtaincallus tissue, a propagule, or a plant containing the genomicmodification. In certain embodiments, a propagule or plant is obtainedby subjecting the plant cell or protoplast to culture systems comprisingsuitable amounts of plant nutrients, vitamins, phytohormones, and thelike that result in regeneration of the propagule or plant. Conditionsfor induction of callus and regeneration of plants and propagules fromplant cells and protoplasts of a variety of plant species disclosed inthe patent literature (US20170145430; US20090038025; US20140173780; eachof which are specifically incorporated herein by reference in theirentireties) and non-patent literature (e.g., S Roest, L J W Gilissen,1989, Acta botanica neerlandica, 38(1):1-23; Bhaskaran and Smith, CropSci. 30(6):1328-1337; Ikeuchi et al., Development, 2016, 143: 1442-1451)can be adapted for use obtaining callus, plant propagules, and plantsfrom the plant cells and protoplasts disclosed herein. Propagules thatcan be obtained include buds, bulbs, corms, rhizomes, stolons, shoots,roots, stems, tubers, or cuttings thereof; as well as seeds, pollen,megaspores, and the like.

In embodiments, the culture, methods, systems, and/or compositionsprovided herein includes plant cells or plant protoplasts obtained fromor located in any monocot or dicot plant species of interest, forexample, row crop plants, fruit-producing plants and trees, vegetables,trees, and ornamental plants including ornamental flowers, shrubs,trees, groundcovers, and turf grasses. In certain embodiments, theculture, methods, systems, and/or compositions provided herein includesplant cells or plant protoplasts obtained from alfalfa (Medicagosativa), almonds (Prunus dulcis), apples (Malus×domestica), apricots(Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P. sibirica),asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Hordeumvulgare), beans (Phaseolus spp.), blueberries and cranberries (Vacciniumspp.), cacao (Theobroma cacao), canola and rapeseed or oilseed rape,(Brassica napus), carnation (Dianthus caryophyllus), carrots (Daucuscarota sativus), cassava (Manihot esculentum), cherry (Prunus avium),chickpea (Cider arietinum), chicory (Cichorium intybus), chili peppersand other capsicum peppers (Capsicum annuum, C. frutescens, C. chinense,C. pubescens, C. baccatum), chrysanthemums (Chrysanthemum spp.), coconut(Cocos nucifera), coffee (Coffea spp. including Coffea arabica andCoffea canephora), cotton (Gossypium hirsutum L.), cowpea (Vignaunguiculata), cucumber (Cucumis sativus), currants and gooseberries(Ribes spp.), eggplant or aubergine (Solanum melongena), eucalyptus(Eucalyptus spp.), flax (Linum usitatissumum L.), geraniums (Pelargoniumspp.), grapefruit (Citrus×paradisi), grapes (Vitus spp.) including winegrapes (Vitus vinifera), guava (Psidium guajava), hemp and cannabis(e.g., Cannabis sativa and Cannabis spp.), hops (Humulus lupulus),irises (Iris spp.), lemon (Citrus limon), lettuce (Lactuca sativa),limes (Citrus spp.), maize (Zea mays L.), mango (Mangifera indica),mangosteen (Garcinia mangostana), melon (Cucumis melo), millets (Setariaspp, Echinochloa spp, Eleusine spp, Panicum spp., Pennisetum spp.), oats(Avena sativa), oil palm (Ellis quineensis), olive (Olea europaea),onion (Allium cepa), orange (Citrus sinensis), papaya (Carica papaya),peaches and nectarines (Prunus persica), pear (Pyrus spp.), pea (Pisasativum), peanut (Arachis hypogaea), peonies (Paeonia spp.), petunias(Petunia spp.), pineapple (Ananas comosus), plantains (Musa spp.), plum(Prunus domestica), poinsettia (Euphorbia pulcherrima), Polish canola(Brassica rapa), poplar (Populus spp.), potato (Solanum tuberosum),pumpkin (Cucurbita pepo), rice (Oryza sativa L.), roses (Rosa spp.),rubber (Hevea brasiliensis), rye (Secale cereale), safflower (Carthamustinctorius L), sesame seed (Sesame indium), sorghum (Sorghum bicolor),soybean (Glycine max L.), squash (Cucurbita pepo), strawberries(Fragaria spp., Fragaria×ananassa), sugar beet (Beta vulgaris),sugarcanes (Saccharum spp.), sunflower (Helianthus annus), sweet potato(Ipomoea batatas), tangerine (Citrus tangerina), tea (Camelliasinensis), tobacco (Nicotiana tabacum L.), tomato (Lycopersiconesculentum), tulips (Tulipa spp.), turnip (Brassica rapa rapa), walnuts(Juglans spp. L.), watermelon (Citrulus lanatus), wheat (Tritiumaestivum), or yams (Discorea spp.). In certain embodiments, the culture,methods, systems, and/or compositions provided herein includes plantcells or plant protoplasts obtained from plants that are typicallypropagated through asexual means, e.g., apples (Malus×domestica),apricots (Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P.sibirica), avocado (Persea americana), bananas (Musa spp.), cherry(Prunus avium), grapefruit (Citrus×paradisi), grapes (Vitus spp.)including wine grapes (Vitus vinifera), irises (Iris spp.), lemon(Citrus limon), limes (Citrus spp.), orange (Citrus sinensis), peachesand nectarines (Prunus persica), pear (Pyrus spp.), pineapple (Ananascomosus), plantains (Musa spp.), plum (Prunus domestica), poinsettia(Euphorbia pulcherrima), potato (Solanum tuberosum), roses (Rosa spp.),strawberries (Fragaria spp., Fragaria×ananassa), sugarcanes (Saccharumspp.), sweet potato (Ipomoea batatas), tangerine (Citrus tangerina), tea(Camellia sinensis), yams (Discorea spp.), hops (Humulus lupulus), andhemp and cannabis (Cannabis sativa and Cannabis spp.) and many otherplants and crops that form bulbs, bulbils, tubers, or corms, or whichmay be propagated by cuttings, root divisions, stolons, runners, orpups.

In embodiments, the culture, methods, systems, compositions, or reactionmixtures provided herein can include plant cells or plant protoplaststhat are (a) encapsulated or enclosed in or attached to a polymer (e.g., pectin, agarose, or other polysaccharide) or other support (solid orsemi-solid surfaces or matrices, or particles or nanoparticles); (b)encapsulated or enclosed in or attached to a vesicle or liposome orother fluid compartment; or (c) not encapsulated or enclosed orattached. In certain embodiments, the culture includes plant cells orplant protoplasts in liquid or suspension culture, or cultured in or onsemi-solid or solid media, or in a combination of liquid and solid orsemi-solid media (e. g., plant cells or protoplasts cultured on solidmedium with a liquid medium overlay, or plant cells or protoplastsattached to solid beads or a matrix and grown with a liquid medium). Incertain embodiments, the culture includes plant cells or plantprotoplasts encapsulated in a polymer (e. g., pectin, agarose, or otherpolysaccharide) or other encapsulating material, enclosed in a vesicleor liposome, suspended in a mixed-phase medium (such as an emulsion orreverse emulsion), or embedded in or attached to a matrix or other solidsupport (e. g., beads or microbeads, membranes, or solid surfaces).

Viability of plant cells or plant protoplasts in a culture, method, orsystem provided herein can be determined by various staining techniques,e. g., by staining dead cells or protoplasts with Evans blue,bromophenol blue, methylene blue, or phenosafranin or staining livecells or protoplasts with fluorescein diacetate. Visual examination ofunstained samples usually correlates well with staining results;live/intact protoplasts retain their round shape and appear to have goodturgor pressure, while dead protoplasts are irregularly shaped, smaller,and appear shriveled. In certain embodiments, in addition to increasedcell viability, culture conditions further provide an improved celldivision rate; this can also be observed by, e. g., microscopicobservations or flow cytometric analysis. Viability of cells orprotoplasts in a culture can be expressed as a percentage, i.e., thepercentage of living or viable cells or protoplasts relative to thetotal number of cells or protoplasts in a sample of the culture;viability can further be measured over a time-course and compared amongdifferent culture conditions. In certain embodiments, the viability ofthe protoplasts in the culture is improved, e. g., by at least 10% afterat least about one day of culture time, when compared to the viabilityof protoplasts in control cultures without (i) a relatively highconcentration (such as at least 20 millimolar) of a divalent cation, or(ii) an antioxidant, such as, but not limited to, a low-molecular-weightthiol antioxidant; or (iii) a combination of (i) and (ii); in certainembodiments, the viability of the protoplasts in the culture is improvedby at least 10%, or by at least 15%, or by at least 20%, or by at least25% after at least about one day of culture time, when compared to theviability of protoplasts in control cultures. In specific embodiments,the viability of the protoplasts in the culture, when compared to acontrol plant protoplast culture without (i) at least 40 millimolar Ca′or Mg²⁺; (ii) an antioxidant; or (iii) a combination of (i) and (ii),is:

(a) at least 10% higher after 30 hours' culture;

(b) at least 10% higher after 48 hours' culture;

(c) at least 10% higher after 72 hours' culture; or

(d) at least 10% higher after 96 hours' culture.

In a specific embodiment, the culture includes at least one plant cellor plant protoplast obtained from maize, the culture medium includes atleast 40, 60, 80, or 100 millimolar Ca²⁺, and protoplast viability is atleast 20% higher after 64 hours' culture when compared to a controlplant protoplast culture without at least 40, 60, 80, or 100 millimolarCa²⁺. In a specific embodiment, the culture includes at least one plantcell or plant protoplast obtained from maize, the culture mediumincludes at least 1 millimolar low-molecular-weight thiol (e. g.,glutathione, cysteine, cysteinyl glycine, gamma-glutamyl cysteine,N-acetylcysteine, cysteine, thiocysteine, homocysteine, lipoic acid,dithiothreitol, or a combination of these), and protoplast viability isat least 10% higher after 64 hours' culture when compared to a controlplant protoplast culture without at least 100 millimolar Ca²⁺.

In another aspect, the disclosure provides a method of improvingviability of a plant cell or a plant protoplast in culture, wherein themethod comprises including in the culture conditions (i. e., in at leastone medium used in culture) of the plant cell or plant protoplast: (i) anon-conventionally high concentration (such as at least 30, at least 40,at least 60, at least 80, or at least 100 millimolar) of a divalentcation (e.g., Ca²⁺ and/or Mg²⁺), or (ii) an antioxidant; or (iii) acombination of (i) and (ii). In an embodiment, the method comprisesincluding in the culture conditions of the plant cell or plantprotoplast at least one of: (i) about 40 to about 150 millimolar Ca²⁺and/or Mg²⁺; and (ii) about 0.1 to about 10 millimolarlow-molecular-weight (non-enzymatic) antioxidant, includinglipid-soluble antioxidants and water-soluble antioxidants, for example,low-molecular-weight thiol antioxidants, ascorbic acid, tocopherols,butylated hydroxytoluene, and butylated hydroxyanisole. In anembodiment, the method comprises including in the culture conditions ofthe plant cell or plant protoplast at least one of: (i) at least 40millimolar Ca²⁺ and/or Mg²⁺; and (ii) about 0.1 to about 10 millimolarlow-molecular-weight antioxidant, such as, but not limited to, alow-molecular-weight thiol antioxidant. In an embodiment, the methodcomprises including in the culture conditions of the plant cell or plantprotoplast at least one of (i) at least 40 millimolar Ca²⁺ and/or Mg²⁺;and (ii) at least 1 millimolar low-molecular-weight thiol (e. g.,glutathione, cysteine, cysteinyl glycine, gamma-glutamyl cysteine,N-acetylcysteine, cysteine, thiocysteine, homocysteine, lipoic acid,dithiothreitol, or a combination of these). In some embodiments, themethod comprises culture conditions that further include hypoxia, oroxygen concentrations that are less than normal atmosphericconcentrations, for example, about one-half normal atmospheric oxygenconcentrations or about 5% to about 10% oxygen by volume. In certainembodiments, such conditions additionally result in an improved celldivision rate in the culture. In certain embodiments, the cultureconditions include at least 40 millimolar Ca²⁺ and/or Mg²⁺ in the mediumand further include maintaining the culture under hypoxic conditions(e.g., about 5% to about 10% oxygen by volume). In certain embodiments,the cell division rate of the plant cells or plant protoplasts isimproved by at least 20%, or by at least 50%, or by at least 75%, or byat least 100%. In an embodiment, the culture conditions include at least40 millimolar Ca²⁺ and about one-half normal atmospheric oxygenconcentrations or about 5% to about 10% oxygen by volume, and the celldivision rate of the plant cells or plant protoplasts is improved by atleast 100% (i. e., cell division rate is about twice that observed insimilar cultures grown under or subjected to normal atmospheric oxygenconcentrations).

In embodiments, the method improves the viability of a plant cell orplant protoplast by at least 10% after at least about one day of culturetime, when compared to the viability of plant cells or plant protoplastsin control cultures without (i) a relatively high concentration (such asat least 20 millimolar) of a divalent cation, or (ii) an antioxidant,such as, but not limited to, a low-molecular-weight thiol antioxidant;or (iii) a combination of (i) and (ii); in certain embodiments, themethod improves the viability of the plant cells or plant protoplasts inthe culture by at least 10%, or by at least 15%, or by at least 20%, orby at least 25% after at least about one day of culture time, whencompared to the viability of plant cells or plant protoplasts in controlcultures.

In specific embodiments, the method comprises including in the cultureconditions of the plant cell or plant protoplast at least one of: (i)between or about 40 to about 150 millimolar Ca²⁺ and/or Mg²⁺; and (ii)between or about 0.1 to about 10 millimolar low-molecular-weightantioxidant; or (iii) a combination of (i) and (ii), whereby viabilityof the plant cells or plant protoplasts in the culture is improved by atleast 10%, or by at least 15%, or by at least 20%, or by at least 25%after at least about one day of culture time, when compared to plantcells or plant protoplasts in a control culture without: (i) between orabout 40 to about 150 millimolar Ca²⁺ or Mg²⁺; (ii) between or about 0.1to about 10 millimolar low-molecular-weight antioxidant; or (iii) acombination of (i) and (ii). In specific embodiments, the methodcomprises including in the culture conditions of the plant cell or plantprotoplast at least one of: (i) at least 40 millimolar Ca²⁺ or Mg²⁺; and(ii) between or about 0.1 to about 10 millimolar low-molecular-weightantioxidant; or (iii) a combination of (i) and (ii), whereby viabilityof the plant cells or plant protoplasts in the culture is improved by:

(a) at least 10% over at least 24 hours' culture;

(b) at least 10% over at least 48 hours' culture;

(c) at least 10% over at least 72 hours' culture; or

(d) at least 10% over at least 96 hours' culture,

when compared to plant cells or plant protoplasts in a control culturewithout: (i) at least 40 millimolar Ca²⁺ or Mg²⁺; (ii) between or about0.1 to about 10 millimolar low-molecular-weight antioxidant; or (iii) acombination of (i) and (ii). In specific embodiments, the methodcomprises including in the culture conditions of the plant cell or plantprotoplast at least one of: (i) at least 40 millimolar Ca²⁺ or Mg²⁺; and(ii) between or about 0.1 to about 10 millimolar low-molecular-weightthiol (e. g., glutathione, cysteine, cysteinyl glycine, gamma-glutamylcysteine, N-acetylcysteine, cysteine, thiocysteine, homocysteine, lipoicacid, dithiothreitol, or a combination of these); or (iii) a combinationof (i) and (ii), whereby viability of the plant cells or plantprotoplasts in the culture is improved by:

(a) at least 10% over at least 24 hours' culture;

(b) at least 10% over at least 48 hours' culture;

(c) at least 10% over at least 72 hours' culture; or

(d) at least 10% over at least 96 hours' culture,

when compared to plant cells or plant protoplasts in a control culturewithout: (i) at least 40 millimolar Ca²⁺ or Mg²⁺; (ii) between or about0.1 to about 10 millimolar low-molecular-weight thiol; or (iii) acombination of (i) and (ii). In specific embodiments, the methodcomprises including in the culture conditions of the plant cell or plantprotoplast at least one of: (i) at least 100 millimolar Ca²⁺ and/orMg²⁺; and (ii) at least 1 millimolar low-molecular-weight thiol (e. g.,glutathione, cysteine, cysteinyl glycine, gamma-glutamyl cysteine,N-acetylcysteine, cysteine, thiocysteine, homocysteine, lipoic acid,dithiothreitol, or a combination of these); or (iii) a combination of(i) and (ii), whereby viability of the plant cells or plant protoplastsin the culture is improved by:

(a) at least 10% over at least 24 hours' culture;

(b) at least 10% over at least 48 hours' culture;

(c) at least 10% over at least 72 hours' culture; or

(d) at least 10% over at least 96 hours' culture,

when compared to plant cells or plant protoplasts in a control culturewithout: (i) at least 100 millimolar Ca²⁺ or Mg²⁺; (ii) at least 1millimolar low-molecular-weight thiol; or (iii) a combination of (i) and(ii).

In embodiments, the method further comprises maintaining the culturemedium under hypoxic conditions, e. g., under about one-half normalatmospheric oxygen concentrations or less, for example, at between orabout 5 to about 10% oxygen by volume, at about 5 to about 10% oxygen byvolume; in certain embodiments, such conditions additionally result inan improved cell division rate in the culture. In certain embodiments,the culture conditions include at least 40 millimolar Ca²⁺ and/or Mg²⁺in the medium and further include maintaining the culture under hypoxicconditions. In certain embodiments, the method further comprisesmaintaining the culture medium under between or about 5 to about 10%oxygen by volume. In certain embodiments, the cell division rate of theprotoplasts is improved compared to that of protoplasts in controlcultures without (i) a relatively high concentration (such as at least20 millimolar) of a divalent cation, or (ii) an antioxidant, such as,but not limited to, a low-molecular-weight thiol antioxidant; or (iii)hypoxic conditions; or (iv) any combination of (i), (ii), and (iii). Incertain embodiments, the cell division rate of the plant cells or plantprotoplasts is improved by at least 20%, or by at least 50%, or by atleast 75%, or by at least 100%. In an embodiment, the culture conditionsinclude at least 40 millimolar Ca²⁺ and about one-half normalatmospheric oxygen concentrations, and the cell division rate of theplant cells or plant protoplasts is improved by at least 100% (i. e.,cell division rate is about twice that observed in similar culturesgrown under or subjected to normal atmospheric oxygen concentrations).

In certain embodiments, the plant cells or plant protoplasts in theculture are generally isolated plant cells or plant protoplasts, that isto say, not located in undissociated or intact plant tissues, plantparts, or whole plants. In certain embodiments, however, plant cells inundissociated or intact plant tissues, plant parts, or whole plants arepre-treated with (i) a non-conventionally high concentration (such as atleast 30, at least 40, at least 60, at least 80, or at least 100millimolar) of a divalent cation, or (ii) an antioxidant; or (iii) acombination of (i) and (ii), prior to the plant cells or plantprotoplasts being isolated from the treated plant tissues, plant parts,or whole plants. In an embodiment, plant cells in undissociated orintact plant tissues, plant parts, or whole plants are pre-treated with:(i) between or about 40 to about 150 millimolar Ca²⁺ or Mg²⁺; (ii)between or about 0.1 to about 10 millimolar low-molecular-weightantioxidant; or (iii) a combination of (i) and (ii), prior to the plantcells or plant protoplasts being isolated from the pre-treated planttissues, plant parts, or whole plants, whereby the viability of theisolated plant cells or protoplasts is improved, relative to controlplant cells or plant protoplasts isolated from plant tissues, plantparts, or whole plants not so pre-treated.

In certain embodiments, of the systems, methods, and compositionsdisclosed herein, dissociated plant cells or plant cells inundissociated or intact plant tissues, plant parts, or whole plants aretreated with (i) a non-conventionally high concentration (e.g., 30, 40,or 60, to 80, 100, 120, or 150 millimolar) of a divalent cation (e.g.,Ca²⁺ and/or Mg²⁺); (ii) a reactive oxygen species (ROS) lowering orscavenging agent (e.g., an antioxidant); (iii) a hypoxia inducing agentor condition (e.g. about 5% to about 10% or 12% oxygen by volume); or(iv) any combination of (i)-(iii). In certain embodiments, suchtreatments are made prior to the plant cells or plant protoplasts beingisolated from the treated plant tissues, plant parts, or whole plantsfor use in the systems, methods and compositions disclosed herein. Incertain embodiments, such treatments are made prior to the plant cellsthat are located within the plant being used in the systems, methods andcompositions disclosed herein. In an embodiment, the disassociated plantcells or plant cells in undissociated or intact plant tissues, plantparts, or whole plants are treated with: (i) about 40 to about 150millimolar Ca²⁺ and/or Mg²⁺; (ii) a ROS scavenging agent comprisingabout 0.1 to about 10 millimolar low-molecular-weight antioxidant; (iii)a hypoxia inducing agent or condition (e.g. about 5% to about 10% or 12%oxygen by volume); or (iv) any combination of (i)-(iii)). In certainembodiments, such treatments are made prior to the plant cells or plantprotoplasts being isolated from the pre-treated plant tissues, plantparts, or whole plants for use in the systems, methods and compositionsdisclosed herein. In certain embodiments, such treatments are made priorto the plant cells that are located within the plant being used in thesystems, methods and compositions disclosed herein. In certainembodiments, any of such aforementioned treatments of the plant cellscan be made in the methods or systems provided herein prior, during,and/or after exposure of the plant cells to genome editing molecules.

In embodiments, the culture, methods, system, or composition includesplant cells or plant protoplasts obtained from or located in any plantpart or tissue or callus. In certain embodiments, the culture includesplant cells or plant protoplasts obtained from a plant tissue, wholeplant, intact nodal bud, shoot apex or shoot apical meristem, root apexor root apical meristem, lateral meristem, intercalary meristem,seedling, whole seed, halved seed or other seed fragment, zygoticembryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl,cotyledon, leaf, petiole, stem, tuber, root, callus, or plant cellsuspension. In certain embodiments, the systems, methods, compositions,or cultures include a plant cell located in a plant tissue, whole plant,intact nodal bud, shoot apex or shoot apical meristem, root apex or rootapical meristem, lateral meristem, intercalary meristem, seedling, wholeseed, halved seed or other seed fragment, zygotic embryo, somaticembryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf,petiole, stem, tuber, root, callus, or plant cell suspension. In certainembodiments, the culture includes haploid, diploid, or polyploid plantcells or plant protoplasts, for example, those obtained from a haploid,diploid, or polyploid plant, plant part or tissue, or callus. In certainembodiments, the culture, method, system, or composition includes plantcells or plant protoplasts obtained from or located in any monocot ordicot plant species of interest, for example, row crop plants,fruit-producing plants and trees, vegetables, trees, and ornamentalplants including ornamental flowers, shrubs, trees, groundcovers, andturf grasses; a non-limiting list of plant species of interest isprovided above under the heading “Plant cell and plant protoplastcultures”. In certain embodiments, the culture, method, system, orcomposition includes plant cells or plant protoplasts that are (a)encapsulated or enclosed in or attached to a polymer (e. g., pectin,agarose, or other polysaccharide) or other support (solid or semi-solidsurfaces or matrices, or particles or nanoparticles); (b) encapsulatedor enclosed in or attached to a vesicle or liposome or other fluidcompartment; or (c) not encapsulated or enclosed or attached. In certainembodiments, the culture includes plant cells or plant protoplasts inliquid or suspension culture, or cultured in or on semi-solid or solidmedia, or in a combination of liquid and solid or semi-solid media (e.g., plant cells or protoplasts cultured on solid medium with a liquidmedium overlay, or plant cells or protoplasts attached to solid beads ora matrix and grown with a liquid medium). In certain embodiments, theculture includes plant cells or plant protoplasts encapsulated in apolymer (e. g., pectin, agarose, or other polysaccharide) or otherencapsulating material, enclosed in a vesicle or liposome, suspended ina mixed-phase medium (such as an emulsion or reverse emulsion), orembedded in or attached to a matrix or other solid support (e. g., beadsor microbeads, membranes, or solid surfaces).

A related aspect of the disclosure relates to the plant cell or plantprotoplast or populations thereof having improved viability and/orincreased gene-editing frequencies, provided by the methods disclosedherein. Also provided by the disclosure are compositions derived from orgrown from the plant cell or plant protoplast having improved viabilityand/or increased gene-editing frequencies, provided by the methodsdisclosed herein; such compositions include multiple protoplasts orcells, callus, a somatic embryo, or a regenerated plant, grown from theplant cell or plant protoplast having improved viability and/orincreased gene-editing frequencies.

Plant cells or plant protoplasts having increased gene-editingfrequencies are provided by the systems and methods disclosed herein.Also provided by the disclosure are compositions derived from or grownfrom the plant cell or plant protoplast having increased gene-editingfrequencies, or provided by the systems and methods disclosed herein. Incertain embodiments, such compositions include multiple protoplasts orcells, callus, a somatic embryo. Also provided are a regenerated orotherwise obtained plant, grown from the plant cell or plant protoplasthaving increased gene-editing frequencies. In certain embodiments, wherethe genome modification comprises homology directed repair (HDR) of thegenome, the frequency of HDR is increased by at least 2-fold, forexample, by about 2-, 3-, 4-, or 5-fold to about 10-, 20, 50-, 100-,200-fold, or more in comparison to a control method wherein a controlplant cell is provided with the genome editing molecules but is notexposed to an ROS concentration lowering agent or a hypoxic conditionand/or is not exposed to a high concentration of a divalent cation(e.g., at least 30 millimolar of Ca²⁺ and/or Mg²⁺). Also provided hereinare populations of plant cells or plant protoplasts that are produced bythe systems, methods, or compositions disclosed herein where thepercentage of the plant cells or protoplasts in the populationcomprising the desired genetic modification in a target gene of interestresulting from the activity of the gene editing molecules is increasedin comparison to a control system, method, or composition wherein atleast one of an ROS concentration lowering agent, a hypoxic condition,and/or a high concentration of divalent cation (e.g., Ca²⁺ and/or Mg²⁺)is absent. In certain embodiments, wherein the genome modificationcomprises homology directed repair (HDR) of the genome, the frequency ofplant cells in the population having the desired genetic modification ina target gene of interest resulting from the activity of the geneediting molecules is increased by at least 2-fold, for example, by about2-, 3-, 4-, or 5-fold to about 10-, 20, 50-, 100-, 200-fold, or more incomparison to a population produced by a control method wherein acontrol plant cell is provided with the genome editing molecules but isnot exposed to an ROS concentration lowering agent or a hypoxiccondition and/or is not exposed to a high concentration of divalentcation (e.g., Ca²⁺ and/or Mg²⁺). In certain embodiments, the increasedfrequency of plant cells in the population having the desired geneticmodification in a target gene of interest resulting from the activity ofthe gene editing molecules is an additive or synergistic increase incomparison to a population produced by a control system, method, orcomposition wherein at least one of a ROS concentration lowering agent,a hypoxic condition, and/or a high concentration of divalent cation(e.g., Ca²⁺ and/or Mg²⁺) is absent.

In some embodiments, the method includes the additional step of growingor regenerating a plant from a plant cell or plant protoplast havingimproved viability and/or increased gene-editing frequencies, a targetgene edit, and/or a genome edit as provided by the methods, systems, andcompositions disclosed herein. In certain embodiments, callus isproduced from the plant cell, and plantlets and plants produced fromsuch callus. In other embodiments, whole seedlings or plants are growndirectly from the plant cell without a callus stage. Thus, additionalrelated aspects are directed to whole seedlings and plants grown orregenerated from the plant cell or plant protoplast having improvedviability and/or increased gene-editing frequencies, a target gene edit,or a genome edit, as well as the seeds of such plants, including seedswith a target gene edit or a genome edit. In certain embodiments whereinthe plant cell or plant protoplast having improved viability and/orincreased gene-editing frequencies is subjected to genetic or epigeneticmodification (for example, stable or transient expression of atransgene, gene silencing, epigenetic silencing, or genome editing byuse of genome modification molecules, e. g., an RNA-guided DNA nucleaseand guide RNA), the grown or regenerated plant exhibits a phenotypeassociated with the genetic or epigenetic modification. In certainembodiments, the grown or regenerated plant includes in its genome twoor more genetic or epigenetic modifications that in combination provideat least one phenotype of interest. In certain embodiments, aheterogeneous population of plant cells or plant protoplasts havingimproved viability and/or increased gene-editing frequencies, at leastsome of which include at least one genetic or epigenetic modification,or a target gene edit, and/or genome edit, is provided by the culture,method, system, or composition. Related aspects include a plant having aphenotype of interest associated with the genetic or epigeneticmodification, provided by either regeneration of a plant having thephenotype of interest from a plant cell or plant protoplast selectedfrom the heterogeneous population of plant cells or plant protoplastshaving improved viability and/or increased gene-editing frequencies, orby selection of a plant having the phenotype of interest from aheterogeneous population of plants grown or regenerated from thepopulation of plant cells or plant protoplasts having improved viabilityand/or increased gene-editing frequencies, a target gene edit, and/or agenome edit. Examples of phenotypes of interest include herbicideresistance, improved tolerance of abiotic stress (e. g., tolerance oftemperature extremes, drought, or salt) or biotic stress (e. g.,resistance to bacterial, nematode, or fungal pathogens), improvedutilization of nutrients or water, modified lipid, carbohydrate, orprotein composition, improved flavour or appearance, improved storagecharacteristics (e. g., resistance to bruising, browning, or softening),increased yield, altered morphology (e. g., floral architecture orcolour, plant height, branching, root structure). In an embodiment, aheterogeneous population of plant cells or plant protoplasts havingimproved viability and/or increased gene-editing frequencies (orseedlings or plants grown or regenerated therefrom) is exposed toconditions permitting expression of the phenotype of interest; e. g.,selection for herbicide resistance can include exposing the populationof plant cells or plant protoplasts having improved viability and/orincreased gene-editing frequencies (or seedlings or plants grown orregenerated therefrom) to an amount of herbicide or other substance thatinhibits growth or is toxic, allowing identification and selection ofthose resistant plant cells or plant protoplasts (or seedlings orplants) that survive treatment. Methods for regenerating plants fromprotoplasts, other plant cells, callus, and the like can be adapted frompublished procedures (e.g., Roest and Gilissen, Acta Bot. Neerl., 1989,38(1), 1-23; Bhaskaran and Smith, Crop Sci. 30(6):1328-1337; Ikeuchi etal., Development, 2016, 143: 1442-1451). Also provided are heterogeneouspopulations, arrays, or libraries of such plant cells, plant cellpopulations, plants, succeeding generations or seeds of such plantsgrown or regenerated from the plant cells or plant protoplasts havingimproved viability, target gene edits, and/or genomic edits, parts ofthe plants (including plant parts used in grafting as scions orrootstocks), or products (e. g., fruits or other edible plant parts,cleaned grains or seeds, edible oils, flours or starches, proteins, andother processed products) made from the plants or their seeds.Embodiments include plants grown or regenerated from the plant cells orplant protoplasts having improved viability and/or increasedgene-editing frequencies, target gene edits, and/or genomic edits,wherein the plants contain cells or tissues that do not have a geneticor epigenetic modification, e. g., grafted plants in which the scion orrootstock contains a genetic or epigenetic modification, or chimericplants in which some but not all cells or tissues contain a genetic orepigenetic modification. Plants in which grafting is commonly usefulinclude many fruit trees and plants such as many citrus trees, apples,stone fruit (e. g., peaches, apricots, cherries, and plums), avocados,tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well asvarious ornamental plants such as roses. Grafted plants can be graftsbetween the same or different (generally related) species. Additionalrelated aspects include a hybrid plant provided by crossing a firstplant grown or regenerated from a plant cell or plant protoplast havingimproved viability and/or increased gene-editing frequencies and havingat least one genetic or epigenetic modification, with a second plant,wherein the hybrid plant contains the genetic or epigeneticmodification; also contemplated is seed produced by the hybrid plant.Also envisioned as related aspects are progeny seed and progeny plants,including hybrid seed and hybrid plants, having the regenerated plant asa parent or ancestor. The plant cells and derivative plants and seedsdisclosed herein can be used for various purposes useful to the consumeror grower. The intact plant itself may be desirable, e. g., plants grownas cover crops or as ornamentals. In other embodiments, processedproducts are made from the plant or its seeds, such as extractedproteins, oils, sugars, and starches, fermentation products, animal feedor human food, wood and wood products, pharmaceuticals, and variousindustrial products. Thus, further related aspects of the disclosureinclude a processed or commodity product made from a plant or seed orplant part that is grown or regenerated from a plant cell or plantprotoplast having improved viability and/or increased gene-editingfrequencies, target gene edits, and/or genomic edits, as disclosedherein. Commodity and processed products include, but are not limitedto, harvested leaves, roots, shoots, tubers, stems, fruits, seeds, orother parts of a plant, meals, oils, extracts, fermentation or digestionproducts, crushed, macerated, and/or ground whole grains or seeds of aplant, wood and wood pulp, or any food or non-food product.

Compositions, plant cell cultures, systems and reaction mixturesincluding plant cells or plant protoplasts having improved viabilityand/or increased gene-editing frequencies are useful, e. g., in methodsinvolving genetic engineering or genome editing. In one aspect, theinvention provides a composition including: (a) at least one plant cellor plant protoplast having improved viability, provided by including inthe culture conditions (i. e., in at least one medium used in culture)of the plant cell or plant protoplast: (i) a non-conventionally highconcentration (such as at least 30, at least 40, at least 60, at least80, or at least 100 millimolar) of a divalent cation, or (ii) anantioxidant, such as, but not limited to, a low-molecular-weight thiolantioxidant; or (iii) a combination of (i) and (ii); (b) at least oneeffector or gene editing molecule (e. g., a polynucleotide or a proteinor a combination of both) for inducing a genetic alteration in the plantcell or plant protoplast; and (c) optionally, at least one deliveryagent (such as at least one chemical, enzymatic, or physical agent). Incertain embodiments, the composition is maintained under hypoxicconditions, e. g., at about half of normal atmospheric oxygenconcentrations, or at between or about 5% to about 10% oxygenconcentrations. Effector or genome editing molecules and delivery agentsare described in further detail below. Embodiments include compositions,plant cell cultures, systems, and methods including at least one plantcell or plant protoplast having improved viability and/or increasedgene-editing frequencies and at least one effector or genome editingmolecule such as an RNA guide for an RNA-guided nuclease (or apolynucleotide encoding an RNA guide for an RNA-guided nuclease); anRNA-guided DNA nuclease (or a polynucleotide encoding an RNA-guided DNAnuclease); an RNA-guided nuclease or RNA-guided DNA nuclease and a guideRNA, and optionally a donor template polynucleotide; a sequence-specificendonuclease and a donor template polynucleotide; one or morepolynucleotides encoding an RNA-guided nuclease or RNA-guided DNAnuclease and a guide RNA, and optionally a donor templatepolynucleotide; one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; orany combination thereof; optionally such compositions further include atleast one chemical, enzymatic, or physical delivery agent. In certainembodiments, the at least one plant cell or plant protoplast included inthe composition or reaction mixture is characterized as having viabilityimproved by at least 10%, or by at least 15%, or by at least 20%, or byat least 25% after at least about one day of culture time, when comparedto the viability of plant cells or plant protoplasts from controlcultures without (i) a non-conventionally high concentration (such as atleast 30, at least 40, at least 60, at least 80, or at least 100millimolar) of a divalent cation, or (ii) an antioxidant, such as, butnot limited to, a low-molecular-weight thiol antioxidant; or (iii) acombination of (i) and (ii). In certain embodiments where the cultureconditions further include hypoxia, the at least one plant cell or plantprotoplast included in the composition or reaction mixture ischaracterized as having improved cell division rates, e. g., improved byat least 25%, or by at least 50%, or by at least about 75%, or by atleast about 100%, in comparison to that observed in plant cells orprotoplasts from control cultures not maintained under hypoxicconditions.

Compositions, plant cell cultures, systems, methods, and reactionmixtures including plant cells or plant protoplasts having increasedtarget gene or genome editing frequencies compared to controls areprovided herein. In certain embodiments, the disclosure provides acomposition, system, or method that comprises: (a) at least one plantcell or plant protoplast that is subjected to hypoxic conditions ortreated with an ROS scavenging agent; (b) one or more gene or genomeediting molecules (e.g., a polynucleotide or a protein or a combinationof both); and optionally (c) a non-conventionally high concentration(e.g., 30, 40, or 60, to 80, 100, 120, or 150 millimolar) of a divalentcation (e.g., Ca²⁺ and/or Mg²⁺); for inducing a genetic modification ina target gene of the plant cell or plant protoplast. Such compositions,systems, or methods can also optionally include at least one deliveryagent (such as at least one chemical, enzymatic, or physical agent thatfacilitates polynucleotide entry into a plant cell or protoplast) and/oroptionally include a non-conventionally high concentration (such as atleast 30, at least 40, at least 60, at least 80, or at least 100millimolar; or any of the aforementioned ranges) of a divalent cation(e.g., Mg²⁺ and/or Ca²⁺). In certain embodiments, the plant cell in thesystem, method, or composition is maintained under hypoxic conditions,e.g., at about half of normal atmospheric oxygen concentrations, or atabout 5% to about 12% oxygen concentration by volume. Embodimentsinclude aforementioned systems, plant cell cultures, methods, andcompositions comprising a gene editing molecule such as an RNA guide foran RNA-guided nuclease (or a polynucleotide encoding an RNA guide for anRNA-guided nuclease) and/or an RNA-guided DNA nuclease (or apolynucleotide encoding an RNA-guided DNA nuclease); optionally suchcompositions further include a donor template polynucleotide and/or atleast one chemical, enzymatic, or physical delivery agent that providesfor entry of the gene editing molecule into the plant cell. In certainembodiments of any of the aforementioned compositions, plant cellcultures, systems, methods, and reaction mixtures, the genome editingmolecule(s) comprise: (i) an RNA-guided nuclease and a guide RNA andoptionally a donor template polynucleotide; (ii) a sequence-specificendonuclease and a donor template polynucleotide; (iii) one or morepolynucleotides encoding an RNA-guided nuclease and a guide RNA andoptionally a donor template polynucleotide; (iv) one or morepolynucleotide(s) encoding a sequence-specific endonuclease and a donortemplate polynucleotide; or (v) any combination thereof.

In certain embodiments, the plant cell culture, system, method, orcomposition includes: (a) at least one plant cell or plant protoplasthaving improved viability and/or increased gene-editing frequencies,provided by including in the culture conditions: (i) between about orabout 40 to about 150 millimolar Ca²⁺ or Mg²⁺; or (ii) between about orabout 0.1 to about 10 millimolar low-molecular-weight antioxidant; or(iii) a combination of (i) and (ii); (b) at least one effector or genomeediting molecule for inducing a genetic alteration in the plant cell orplant protoplast; and (c) optionally, at least one chemical, enzymatic,or physical delivery agent. In another embodiment, the compositionincludes: (a) at least one plant cell or plant protoplast havingimproved viability and/or increased gene-editing frequencies, providedby including in the culture conditions: (i) at least 40 millimolar Ca²⁺or Mg²⁺; or (ii) between about or about 0.1 to about 10 millimolarlow-molecular-weight thiol (e. g., glutathione, cysteine, cysteinylglycine, gamma-glutamyl cysteine, N-acetylcysteine, cysteine,thiocysteine, homocysteine, lipoic acid, dithiothreitol, or acombination of these); or (iii) a combination of (i) and (ii); (b) atleast one effector or genome editing molecule for inducing a geneticalteration in the plant cell or plant protoplast; and (c) optionally, atleast one chemical, enzymatic, or physical delivery agent. In anotherembodiment, the composition includes: (a) at least one plant cell orplant protoplast having improved viability and/or increased gene-editingfrequencies, provided by including in the culture conditions: (i) atleast 100 millimolar Ca²⁺ or Mg²⁺; or (ii) at least 1 millimolarlow-molecular-weight thiol (e. g., glutathione, cysteine, cysteinylglycine, gamma-glutamyl cysteine, N-acetylcysteine, cysteine,thiocysteine, homocysteine, lipoic acid, dithiothreitol, or acombination of these); or (iii) a combination of (i) and (ii); (b) atleast one effector or genome editing molecule for inducing a geneticalteration in the plant cell or plant protoplast; and (c) optionally, atleast one chemical, enzymatic, or physical delivery agent. In certainembodiments of any of the aforementioned compositions, plant cellcultures, systems, methods, and reaction mixtures, the genome editingmolecule(s) comprise: (i) an RNA-guided nuclease and a guide RNA andoptionally a donor template polynucleotide; (ii) a sequence-specificendonuclease and a donor template polynucleotide; (iii) one or morepolynucleotides encoding an RNA-guided nuclease and a guide RNA andoptionally a donor template polynucleotide; (iv) one or morepolynucleotide(s) encoding a sequence-specific endonuclease and a donortemplate polynucleotide; or (v) any combination thereof.

In specific embodiments, the plant cell culture, system, methods, orcomposition includes: (a) at least one plant cell or plant protoplasthaving improved viability and/or increased gene-editing frequencies,provided by including in the culture conditions: (i) between about orabout 40 to about 150 millimolar Ca²⁺ or Mg²⁺; and (ii) between about orabout 0.1 to about 10 millimolar low-molecular-weight antioxidant; or(iii) a combination of (i) and (ii), whereby viability of the plantcells or plant protoplasts in the culture is improved by at least 10%,or by at least 15%, or by at least 20%, or by at least 25% after atleast about one day of culture time, when compared to plant cells orplant protoplasts in a control culture without: (i) between about orabout 40 to about 150 millimolar Ca²⁺ or Mg²⁺; (ii) between about orabout 0.1 to about 10 millimolar low-molecular-weight antioxidant; or(iii) a combination of (i) and (ii); (b) at least one effector or genomeediting molecule for inducing a genetic alteration in the plant cell orplant protoplast; and (c) optionally, at least one chemical, enzymatic,or physical delivery agent. In certain embodiments of any of theaforementioned compositions, plant cell cultures, systems, methods, andreaction mixtures, the genome editing molecule(s) comprise: (i) anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (ii) a sequence-specific endonuclease and a donortemplate polynucleotide; (iii) one or more polynucleotides encoding anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (iv) one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof.

In specific embodiments, the plant cell culture, system, method, orcomposition includes: (a) at least one plant cell or plant protoplasthaving improved viability and/or increased gene-editing frequencies,provided by including in the culture conditions: (i) at least 40millimolar Ca²⁺ or Mg²⁺; and (ii) between about or about 0.1 to about 10millimolar low-molecular-weight antioxidant; or (iii) a combination of(i) and (ii), whereby viability of the plant cells or plant protoplastsin the culture is improved by:

(a) at least 10% over at least 24 hours' culture;

(b) at least 10% over at least 48 hours' culture;

(c) at least 10% over at least 72 hours' culture; or

(d) at least 10% over at least 96 hours' culture,

when compared to plant cells or plant protoplasts in a control culturewithout: (i) at least 40 millimolar Ca²⁺ or Mg²⁺; (ii) between about orabout 0.1 to about 10 millimolar low-molecular-weight antioxidant; or(iii) a combination of (i) and (ii); (b) at least one effector or genomeediting molecule for inducing a genetic alteration in the plant cell orplant protoplast; and (c) optionally, at least one chemical, enzymatic,or physical delivery agent. In specific embodiments, the compositionincludes: (a) at least one plant cell or plant protoplast havingimproved viability and/or increased gene-editing frequencies, providedby including in the culture conditions: (i) at least 40 millimolar Ca²⁺or Mg²⁺; and (ii) between or about 0.1 to about 10 millimolarlow-molecular-weight thiol (e. g., glutathione, cysteine, cysteinylglycine, gamma-glutamyl cysteine, N-acetylcysteine, cysteine,thiocysteine, homocysteine, lipoic acid, dithiothreitol, or acombination of these); or (iii) a combination of (i) and (ii), wherebyviability of the plant cells or plant protoplasts in the culture isimproved by:

(a) at least 10% over at least 24 hours' culture;

(b) at least 10% over at least 48 hours' culture;

(c) at least 10% over at least 72 hours' culture; or

(d) at least 10% over at least 96 hours' culture,

when compared to plant cells or plant protoplasts in a control culturewithout: (i) at least 40 millimolar Ca²⁺ or Mg²⁺; (ii) between about orabout 0.1 to about 10 millimolar low-molecular-weight thiol; or (iii) acombination of (i) and (ii); (b) at least one effector or genome editingmolecule for inducing a genetic alteration in the plant cell or plantprotoplast; and (c) optionally, at least one chemical, enzymatic, orphysical delivery agent. In specific embodiments, the compositionincludes: (a) at least one plant cell or plant protoplast havingimproved viability and/or increased gene-editing frequencies, providedby including in the culture conditions: (i) at least 100 millimolar Ca²⁺or Mg²⁺; and (ii) at least 1 millimolar low-molecular-weight thiol (e.g., glutathione, cysteine, cysteinyl glycine, gamma-glutamyl cysteine,N-acetylcysteine, cysteine, thiocysteine, homocysteine, lipoic acid,dithiothreitol, or a combination of these); or (iii) a combination of(i) and (ii), whereby viability of the plant cells or plant protoplastsin the culture is improved by:

(a) at least 10% over at least 24 hours' culture;

(b) at least 10% over at least 48 hours' culture;

(c) at least 10% over at least 72 hours' culture; or

(d) at least 10% over at least 96 hours' culture,

when compared to plant cells or plant protoplasts in a control culturewithout: (i) at least 100 millimolar Ca²⁺ or Mg²⁺; (ii) at least 1millimolar low-molecular-weight thiol; or (iii) a combination of (i) and(ii); (b) at least one effector molecule for inducing a geneticalteration in the plant cell or plant protoplast (e.g., genome editingmolecules for inducing a genetic modification); and (c) optionally, atleast one chemical, enzymatic, or physical delivery agent. In variousembodiments (such as, but not limited to those described above), the atleast one effector or genome editing molecule for inducing a geneticalteration in the plant cell or plant protoplast is selected from thegroup consisting of: (i) a polynucleotide selected from the groupconsisting of an RNA guide for an RNA-guided nuclease, a DNA encoding anRNA guide for an RNA-guided nuclease; (ii) a nuclease selected from thegroup consisting of an RNA-guided nuclease, an RNA-guided DNAendonuclease, a type II Cas nuclease, a Cas9, a type V Cas nuclease, aCpfl, a CasY, a CasX, a C2c1, a C2c3, an engineered nuclease, acodon-optimized nuclease, a zinc-finger nuclease (ZFN), a transcriptionactivator-like effector nuclease (TAL-effector nuclease), Argonaute, ameganuclease or engineered meganuclease; or (iii) a polynucleotideencoding one or more nucleases capable of effecting site-specificalteration of a target nucleotide sequence; and/or (iv) a donor templatepolynucleotide. In various embodiments (such as, but not limited tothose described above), the at least one delivery agent is selected fromthe group consisting of solvents, fluorocarbons, glycols or polyols,surfactants; primary, secondary, or tertiary amines and quaternaryammonium salts; organosilicone surfactants; lipids, lipoproteins,lipopolysaccharides; acids, bases, caustic agents; peptides, proteins,or enzymes; cell-penetrating peptides; RNase inhibitors; cationicbranched or linear polymers; dendrimers; counter-ions, amines orpolyamines, osmolytes, buffers, and salts; polynucleotides; transfectionagents; antibiotics; chelating agents such as ammonium oxalate, EDTA,EGTA, or cyclohexane diamine tetraacetate; non-specific DNAdouble-strand-break-inducing agents; and antioxidants; particles ornanoparticles, magnetic particles or nanoparticles, abrasive orscarifying agents, needles or microneedles, matrices, and grids. Incertain embodiments, the culture, method, system, or compositionincludes (a) at least one plant cell or plant protoplast having improvedviability and/or increased gene-editing frequencies, provided byincluding in the culture conditions: (i) between about or about 40 toabout 150 millimolar Ca²⁺ or Mg²⁺; and (ii) between about or about 0.1to about 10 millimolar low-molecular-weight antioxidant; or (iii) acombination of (i) and (ii), whereby viability of the plant cells orplant protoplasts in the culture is improved by at least 10%, or by atleast 15%, or by at least 20%, or by at least 25% after at least aboutone day of culture time, when compared to plant cells or plantprotoplasts in a control culture without: (i) between about or about 40to about 150 millimolar Ca²⁺ or Mg²⁺; (ii) between about or about 0.1 toabout 10 millimolar low-molecular-weight antioxidant; or (iii) acombination of (i) and (ii); (b) a Cas9, a Cpfl, a CasY, a CasX, a C2c1,or a C2c3 nuclease; (c) at least one guide RNA; and (d) optionally, atleast one chemical, enzymatic, or physical delivery agent. In certainembodiments, the composition includes (a) at least one plant cell orplant protoplast having improved viability and/or increased gene-editingfrequencies, provided by including in the culture conditions: (i)between or about 40 to about 150 millimolar Ca²⁺ or Mg²⁺; and (ii)between about or about 0.1 to about 10 millimolar low-molecular-weightantioxidant; or (iii) a combination of (i) and (ii), whereby viabilityof the plant cells or plant protoplasts in the culture is improved by atleast 10%, or by at least 15%, or by at least 20%, or by at least 25%after at least about one day of culture time, when compared to plantcells or plant protoplasts in a control culture without: (i) betweenabout or about 40 to about 150 millimolar Ca²⁺ or Mg²⁺; (ii) betweenabout or about 0.1 to about 10 millimolar low-molecular-weightantioxidant; or (iii) a combination of (i) and (ii); (b) at least oneribonucleoprotein including a CRISPR nuclease and a guide RNA; and (c)optionally, at least one chemical, enzymatic, or physical deliveryagent. In certain embodiments of any of the aforementioned compositions,plant cell cultures, systems, methods, and reaction mixtures, the genomeediting molecule(s) comprise: (i) an RNA-guided nuclease and a guide RNAand optionally a donor template polynucleotide; (ii) a sequence-specificendonuclease and a donor template polynucleotide; (iii) one or morepolynucleotides encoding an RNA-guided nuclease and a guide RNA andoptionally a donor template polynucleotide; (iv) one or morepolynucleotide(s) encoding a sequence-specific endonuclease and a donortemplate polynucleotide; or (v) any combination thereof.

Embodiments of the plant cell cultures, methods, systems, andcompositions as described in the immediately preceding paragraphsfurther include those wherein the culture is maintained under hypoxicconditions, e. g., under about one-half normal atmospheric oxygenconcentrations or less, for example, at between about or about 5 toabout 10%, 11%, or 12% oxygen by volume or any of the other hypoxicconditions disclosed herein. In certain embodiments, such conditionsadditionally result in an improved cell division rate in the culture. Incertain embodiments, the cell division rate of the cells or protoplastsin the composition is improved by at least 10%, or by at least 15%, orby at least 20%, or by at least 25%, or by at least 50%, or by at least75%, or by at least 100%, or by at least 2-fold, compared to that ofcells or protoplasts in control cultures that are similar except thatthey are not maintained under hypoxic conditions. In certainembodiments, the plant cells (e.g., plant protoplasts) are exposed tothe hypoxic conditions about 5, 10, 15, 30, or 45 minutes to about 60,75, 90, or 120 minutes after exposure to the gene-editing molecules.

In a related aspect, the disclosure provides arrangements of plant cellsor plant protoplasts having improved viability and/or increasedgene-editing frequencies as provided by the methods and cultureconditions described herein, such as arrangements of plant cells orplant protoplasts convenient for screening purposes or forhigh-throughput and/or multiplex gene editing experiments. Plant cellcultures, compositions, and systems provided herein can be used in thearrangements. Methods provided herein can also be practiced in sucharrangements. In an embodiment, the disclosure provides a pooledarrangement of multiple plant cells or plant protoplasts having improvedviability and/or increased gene-editing frequencies, provided byincluding in the culture conditions of the plant cell or plantprotoplast (i) a relatively high concentration (such as at least 20millimolar) of a divalent cation, or (ii) an antioxidant, such as, butnot limited to, a low-molecular-weight thiol antioxidant; or (iii) acombination of (i) and (ii); a specific embodiment is such a pooledarrangement of multiple plant cells or plant protoplasts having improvedviability and/or increased gene-editing frequencies, further includingat least one effector or genome editing molecule (e. g., an RNA-guidedDNA nuclease, at least one guide RNA, or a ribonucleoprotein includingboth an RNA-guided DNA nuclease and at least one guide RNA), andoptionally at least one chemical, enzymatic, or physical delivery agent.In certain embodiments, the disclosure provides an arrangement ofmultiple plant cells or plant protoplasts: (a) a plant cell subjected toa hypoxic condition, or treated with a reactive oxygen species (ROS)scavenging agent, or both subjected to the hypoxic condition and treatedwith the ROS scavenging agent; and/or (b) a non-conventionally highconcentration (e.g., 30, 40, or 60, to 80, 100, 120, or 150 millimolar)of a divalent cation (e.g., Ca²⁺ and/or Mg²⁺); and (c) genome editingmolecule(s). In certain embodiments, the arrangements of plant cells canfurther comprise at least one chemical, enzymatic, or physical deliveryagent. In another embodiment, the disclosure provides an array includinga plurality of containers, each including at least one plant cell orplant protoplast having improved viability and/or increased gene-editingfrequencies, provided by including in the culture conditions of theplant cell or plant protoplast (i) a relatively high concentration (suchas at least 20 millimolar) of a divalent cation, or (ii) an antioxidant,such as, but not limited to, a low-molecular-weight thiol antioxidant;or (iii) a combination of (i) and (ii). In an embodiment, the disclosureprovides arrangements of plant cells or plant protoplasts havingimproved viability and/or increased gene-editing frequencies, whereinthe plant cells or plant protoplasts are in an arrayed format, forexample, in multi-well plates, encapsulated or enclosed in vesicles,liposomes, or droplets (useful, (e. g., in a microfluidics device), orattached discretely to a matrix or to discrete particles or beads; aspecific embodiment is such an arrangement of multiple plant cells orplant protoplasts having improved viability and/or increasedgene-editing frequencies provided in an arrayed format, furtherincluding at least one effector or genome editing molecule (e. g., anRNA-guided DNA nuclease, at least one guide RNA, or a ribonucleoproteinincluding both an RNA-guided DNA nuclease and at least one guide RNA),which may be different for at least some locations on the array or evenfor each location on the array, and optionally at least one chemical,enzymatic, or physical delivery agent. In certain embodiments of any ofthe aforementioned compositions, plant cell cultures, systems, methods,and reaction mixtures involving such arrangements or arrays, the genomeediting molecule(s) comprise: (i) an RNA-guided nuclease and a guide RNAand optionally a donor template polynucleotide; (ii) a sequence-specificendonuclease and a donor template polynucleotide; (iii) one or morepolynucleotides encoding an RNA-guided nuclease and a guide RNA andoptionally a donor template polynucleotide; (iv) one or morepolynucleotide(s) encoding a sequence-specific endonuclease and a donortemplate polynucleotide; or (v) any combination thereof.

In the systems and methods provided herein, plant cells can be exposedor subjected to gene editing molecules and a reactive oxygen species(ROS) concentration lowering agent and/or hypoxic condition in anytemporal order. In certain embodiments, the gene editing molecules and areactive oxygen species (ROS) concentration lowering agent and/orhypoxic condition are provided simultaneously. In other embodiments, thegene editing molecules are provided simultaneously after a reactiveoxygen species (ROS) concentration lowering agent and/or hypoxiccondition is provided. In other embodiments, the gene editing moleculesare provided and a reactive oxygen species (ROS) concentration loweringagent and/or a hypoxic condition is subsequently provided. In summary,the genome editing molecules can be provided to a plant cell eitherprevious to, concurrently with, or subsequent to exposing the plant cellto: (i) a hypoxic condition, a reactive oxygen species (ROS)concentration lowering agent, or combination thereof.

Effector Molecules: Effector molecules (e.g., gene editing molecules) ofuse in the compositions and reaction mixtures provided herein includemolecules capable of introducing a double-strand break (“DSB”) indouble-stranded DNA, such as in genomic DNA or in a target gene locatedwithin the genomic DNA as well as accompanying guide RNA or donortemplate polynucleotides. Examples of such gene editing moleculesinclude: (a) a nuclease selected from the group consisting of anRNA-guided nuclease, an RNA-guided DNA endonuclease, a type II Casnuclease, a Cas9, a type V Cas nuclease, a Cpfl, a CasY, a CasX, a C2c1,a C2c3, an engineered nuclease, a codon-optimized nuclease, azinc-finger nuclease (ZFN), a transcription activator-like effectornuclease (TAL-effector nuclease), an Argonaute, and a meganuclease orengineered meganuclease; (b) a polynucleotide encoding one or morenucleases capable of effecting site-specific alteration (such asintroduction of a DSB) of a target nucleotide sequence; and (c) a guideRNA (gRNA) for an RNA-guided nuclease, or a DNA encoding a gRNA for anRNA-guided nuclease; and (d) donor template polynucleotides.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR-associated) systems, or CRISPR systems, are adaptive defensesystems originally discovered in bacteria and archaea. CRISPR systemsuse RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases(e. g., Cas9 or Cpfl) to cleave foreign DNA. In a typical CRISPR/Cassystem, a Cas endonuclease is directed to a target nucleotide sequence(e. g., a site in the genome that is to be sequence-edited) bysequence-specific, non-coding “guide RNAs” that target single- ordouble-stranded DNA sequences. In microbial hosts, CRISPR loci encodeboth Cas endonucleases and “CRISPR arrays” of the non-coding RNAelements that determine the specificity of the CRISPR-mediated nucleicacid cleavage.

Three classes (I-III) of CRISPR systems have been identified across awide range of bacterial hosts and can be adapted for use in the plantcell cultures, systems, methods, and compositions provided herein. Thewell characterized class II CRISPR systems use a single Cas endonuclease(rather than multiple Cas proteins). One class II CRISPR system includesa type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and atrans-activating crRNA (“tracrRNA”). The crRNA contains a “guide RNA”,typically a 20-nucleotide RNA sequence that corresponds to (i. e., isidentical or nearly identical to, or alternatively is complementary ornearly complementary to) a 20-nucleotide target DNA sequence. The crRNAalso contains a region that binds to the tracrRNA to form a partiallydouble-stranded structure which is cleaved by RNase III, resulting in acrRNA/tracrRNA hybrid. The crRNA/tracrRNA hybrid then directs the Cas9endonuclease to recognize and cleave the target DNA sequence.

The target DNA sequence must generally be adjacent to a “protospaceradjacent motif” (“PAM”) that is specific for a given Cas endonuclease;however, PAM sequences are short and relatively non-specific, appearingthroughout a given genome. CRISPR endonucleases identified from variousprokaryotic species have unique PAM sequence requirements; examples ofPAM sequences include 5′-NGG (Streptococcus pyogenes), 5′-NNAGAA (SEQ IDNO:1; Streptococcus thermophilus CRISPR1), 5′-NGGNG (SEQ ID NO:2;Streptococcus thermophilus CRISPR3), 5′-NNGRRT (SEQ ID NO:3); or5′-NNGRR (SEQ ID NO:4; Staphylococcus aureus Cas9, SaCas9), and5′-NNNGATT (SEQ ID NO:5: Neisseria meningitidis). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g.,5′-NGG, and perform blunt-end cleaving of the target DNA at a location 3nucleotides upstream from (5′ from) the PAM site.

Another class II CRISPR system that can be adapted for use in the plantcell cultures, systems, methods, and compositions provided hereinincludes the type V endonuclease Cpfl, which is a smaller endonucleasethan is Cas9; examples include AsCpfl (from Acidaminococcus sp.) andLbCpfl (from Lachnospiraceae sp.). Cpfl-associated CRISPR arrays areprocessed into mature crRNAs without the requirement of a tracrRNA; inother words, a Cpfl system requires only the Cpfl nuclease and a crRNAto cleave the target DNA sequence. Cpfl endonucleases, are associatedwith T-rich PAM sites, e. g., 5′-TTN. Cpfl can also recognize a 5′-CTAPAM motif. Cpfl cleaves the target DNA by introducing an offset orstaggered double-strand break with a 4- or 5-nucleotide 5′ overhang, forexample, cleaving a target DNA with a 5-nucleotide offset or staggeredcut located 18 nucleotides downstream from (3′ from) from the PAM siteon the coding strand and 23 nucleotides downstream from the PAM site onthe complimentary strand; the 5-nucleotide overhang that results fromsuch offset cleavage allows more precise genome editing by DNA insertionby homologous recombination than by insertion at blunt-end cleaved DNA.See, e. g., Zetsche et al. (2015) Cell, 163:759-771. Other CRISPRnucleases useful in methods, systems, and compositions of the disclosureinclude C2c1 and C2c3 (see Shmakov et al. (2015) Mol. Cell, 60:385-397).Like other CRISPR nucleases, C2c1 from Alicyclobacillus acidoterrestris(AacC2c1; amino acid sequence with accession ID T0D7A2, depositedon-line at www[dot]ncbi[dot]nlm[dot]nih[dot]gov/protein/1076761101)requires a guide RNA and PAM recognition site; C2c1 cleavage results ina staggered seven-nucleotide DSB in the target DNA (see Yang et al.(2016) Cell, 167:1814-1828.e12) and is reported to have high mismatchsensitivity, thus reducing off-target effects (see Liu et al. (2016)Mol. Cell, available on line atdx[dot]doi[dot]org/10[dot]1016/j[dot]molce1[dot]2016 [dot]11.040).Another CRISPR nuclease, Campylobacter jejuni-derived Cas9 (CjCas9), isonly 984 amino acids in length (considerably smaller than, for example,S. pyogenes Cas9 at 1368 amino acids or S. aureus Cas9 at 1053 aminoacids); CjCas9 also requires a guide RNA (reported to be optimal for a22-nucleotide target sequence) and a PAM recognition site (reported tobe 5′-NNNNACAC (SEQ ID NO:6), 5′-NNNNRY (SEQ ID NO:7), 5′-NNNNACA (SEQID NO:8), or 5′-NNNNRYAC (SEQ ID NO:9), where R is a purine and Y is apyrimidine); see Kim et al. (2017) Nature Communications, 8:14500(doi:10.1038/ncomms14500). Yet other CRISPR nucleases include nucleasesidentified from the genomes of uncultivated microbes, such as CasX andCasY (e. g., a CRISPR-associated protein CasY from an unculturedParcubacteria group bacterium, amino acid sequence with accession IDAPG80656, deposited on-line atwww[dot]ncbi[dot]nlm[dot]nih[dot]gov/protein/APG80656.1]); see Bursteinet al. (2016) Nature, doi:10.1038/nature21059.

CRISPR-type genome editing has value in various aspects of agricultureresearch and development and can be adapted for use in the systems,methods, and compositions provided herein in several ways. CRISPRelements, i. e., CRISPR endonucleases and CRISPR single-guide RNAs, areuseful in effecting genome editing without remnants of the CRISPRelements or selective genetic markers occurring in progeny. In certainembodiments, the CRISPR elements are provided directly to the systems,methods, and compositions as isolated molecules, as isolated orsemi-purified products of a cell free synthetic process (e.g., in vitrotranslation), or as isolated or semi-purified products of in acell-based synthetic process (e.g., such as in a bacterial or other celllysate. Alternatively, genome-inserted CRISPR elements are useful inplant lines adapted for multiplex genetic screening and breeding. Forinstance, a plant species can be created to express one or more of aCRISPR endonuclease such as a Cas9- or a Cpfl-type endonuclease orcombinations with unique PAM recognition sites. Cpfl endonuclease andcorresponding guide RNAs and PAM sites are disclosed in US PatentApplication Publication 2016/0208243 A1, which is incorporated herein byreference for its disclosure of DNA encoding Cpfl endonucleases andguide RNAs and PAM sites. Introduction of one or more of a wide varietyof CRISPR guide RNAs that interact with CRISPR endonucleases integratedinto a plant genome or otherwise provided to a plant is useful forgenetic editing for providing desired phenotypes or traits, for traitscreening, or for trait introgression. Multiple endonucleases can beprovided in expression cassettes with the appropriate promoters to allowmultiple genome editing in a spatially or temporally separated fashionin either in chromosome DNA or episome DNA.

Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specificDNA sequences targeted by a gRNA, a number of CRISPR endonucleaseshaving modified functionalities are available and can be used in thesystems, methods, and compositions provided herein. For example: (1) a“nickase” version of Cas9 generates only a single-strand break; (2) acatalytically inactive Cas9 (“dCas9”) does not cut the target DNA butinterferes with transcription; dCas9 can further be fused with arepressor peptide; (3) a catalytically inactive Cas9 (“dCas9”) fused toan activator peptide can activate or increase gene expression; (4) acatalytically inactive Cas9 (dCas9) fused to FokI nuclease(“dCas9-FokI”) can be used to generate DSBs at target sequenceshomologous to two gRNAs. See, e. g., the numerous CRISPR/Cas9 plasmidsdisclosed in and publicly available from the Addgene repository(Addgene, 75 Sidney St., Suite 550A, Cambridge, Mass. 02139;addgene[dot]org/crispr/). A “double nickase” Cas9 that introduces twoseparate double-strand breaks, each directed by a separate guide RNA, isdescribed as achieving more accurate genome editing by Ran et al. (2013)Cell, 154:1380-1389.

CRISPR technology for editing the genes of eukaryotes is disclosed in USPatent Application Publications 2016/0138008A1 and US2015/0344912A1, andin U.S. Pat. Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233,8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814,8,795,965, and 8,906,616. Cpfl endonuclease and corresponding guide RNAsand PAM sites are disclosed in US Patent Application Publication2016/0208243 A1. Other CRISPR nucleases useful for editing genomesinclude C2c1 and C2c3 (see Shmakov et al. (2015) Mol. Cell, 60:385-397)and CasX and CasY (see Burstein et al. (2016) Nature,doi:10.1038/nature21059). Plant RNA promoters for expressing CRISPRguide RNA and plant codon-optimized CRISPR Cas9 endonuclease aredisclosed in International Patent Application PCT/US2015/018104(published as WO 2015/131101 and claiming priority to U.S. ProvisionalPatent Application 61/945,700). Methods of using CRISPR technology forgenome editing in plants are disclosed in US Patent ApplicationPublications US 2015/0082478A1 and US 2015/0059010A1 and inInternational Patent Application PCT/US2015/038767 A1 (published as WO2016/007347 and claiming priority to U.S. Provisional Patent Application62/023,246). All of the patent publications referenced in this paragraphare incorporated herein by reference in their entirety.

For the purposes of gene editing, CRISPR arrays can be designed tocontain one or multiple guide RNA sequences corresponding to a desiredtarget DNA sequence; see, for example, Cong et al. (2013) Science,339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. At least16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNAcleavage to occur; for Cpfl at least 16 nucleotides of gRNA sequence areneeded to achieve detectable DNA cleavage and at least 18 nucleotides ofgRNA sequence were reported necessary for efficient DNA cleavage invitro; see Zetsche et al. (2015) Cell, 163:759-771. In practice, guideRNA sequences are generally designed to have a length of between orabout 17-24 nucleotides (frequently 19, 20, or 21 nucleotides) and exactcomplementarity (i. e., perfect base-pairing) to the targeted gene ornucleic acid sequence; guide RNAs having less than 100% complementarityto the target sequence can be used (e. g., a gRNA with a length of 20nucleotides and between or about 1-4 mismatches to the target sequence)but can increase the potential for off-target effects. The design ofeffective guide RNAs for use in plant genome editing is disclosed in USPatent Application Publication 2015/0082478 A1, the entire specificationof which is incorporated herein by reference. More recently, efficientgene editing has been achieved using a chimeric “single guide RNA”(“sgRNA”), an engineered (synthetic) single RNA molecule that mimics anaturally occurring crRNA-tracrRNA complex and contains both a tracrRNA(for binding the nuclease) and at least one crRNA (to guide the nucleaseto the sequence targeted for editing); see, for example, Cong et al.(2013) Science, 339:819-823; Xing et al. (2014) BMC Plant Biol.,14:327-340. Chemically modified sgRNAs have been demonstrated to beeffective in genome editing; see, for example, Hendel et al. (2015)Nature Biotechnol., 985-991.

In embodiments, the guide RNA (gRNA) has a sequence of between or about16-24 nucleotides in length (e. g., 16, 17, 18, 19, 20, 21, 22, 23, or24 nucleotides in length). Specific embodiments include gRNAs of 19, 20,or 21 nucleotides in length and having 100% complementarity to thetarget nucleotide sequence. In many embodiments the gRNA has exactcomplementarity (i. e., perfect base-pairing) to the target nucleotidesequence; in certain other embodiments the gRNA has less than 100%complementarity to the target nucleotide sequence. The design ofeffective gRNAs for use in plant genome editing is disclosed in USPatent Application Publication 2015/0082478 A1, the entire specificationof which is incorporated herein by reference. In certain embodimentswhere multiple gRNAs are employed, the multiple gRNAs can be deliveredseparately (as separate RNA molecules or encoded by separate DNAmolecules) or in combination, e. g., as an RNA molecule containingmultiple gRNA sequences or as a DNA molecule encoding an RNA moleculecontaining multiple gRNA sequences; see, for example, US PatentApplication Publication 2016/0264981 A1, the entire specification ofwhich is incorporated herein by reference, which discloses RNA moleculesincluding multiple RNA sequences (such as gRNA sequences) separated bytRNA cleavage sequences. Efficient Cas9-mediated gene editing has beenachieved using a chimeric “single guide RNA” (“sgRNA”), an engineered(synthetic) single RNA molecule that mimics a naturally occurringcrRNA-tracrRNA complex and contains both a tracrRNA (for binding thenuclease) and at least one crRNA (to guide the nuclease to the sequencetargeted for editing).

Thus, in certain embodiments wherein the nuclease is a Cas9-typenuclease, the gRNA can be provided as a polynucleotide compositionincluding: (a) a CRISPR RNA (crRNA) that includes the gRNA together witha separate tracrRNA, or (b) at least one polynucleotide that encodes acrRNA and a tracrRNA (on a single polynucleotide or on separatepolynucleotides), or (c) at least one polynucleotide that is processedinto one or more crRNAs and a tracrRNA. In other embodiments wherein thenuclease is a Cas9-type nuclease, the gRNA can be provided as apolynucleotide composition including a CRISPR RNA (crRNA) that includesthe gRNA, and the required tracrRNA is provided in a separatecomposition or in a separate step, or is otherwise provided to the cell(for example, to a plant cell or plant protoplast that stably ortransiently expresses the tracrRNA from a polynucleotide encoding thetracrRNA). In other embodiments wherein the nuclease is a Cas9-typenuclease, the gRNA can be provided as a polynucleotide compositioncomprising: (a) a single guide RNA (sgRNA) that includes the gRNA, or(b) a polynucleotide that encodes a sgRNA, or (c) a polynucleotide thatis processed into a sgRNA. Cpfl-mediated gene editing does not require atracrRNA; thus, in certain embodiments wherein the nuclease is aCpfl-type nuclease, the gRNA is provided as a polynucleotide compositioncomprising (a) a CRISPR RNA (crRNA) that includes the gRNA, or (b) apolynucleotide that encodes a crRNA, or (c) a polynucleotide that isprocessed into a crRNA. In certain embodiments, the gRNA-containingcomposition optionally includes an RNA-guided nuclease, or apolynucleotide that encodes the RNA-guided nuclease. In otherembodiments, an RNA-guided nuclease or a polynucleotide that encodes theRNA-guided nuclease is provided in a separate step. In some embodimentsof the method, a gRNA is provided to a cell (e. g., a plant cell orplant protoplast) that includes an RNA-guided nuclease or apolynucleotide that encodes an RNA-guided nuclease, e. g., an RNA-guidednuclease selected from the group consisting of an RNA-guided DNAendonuclease, a type II Cas nuclease, a Cas9, a type V Cas nuclease, aCpfl, a CasY, a CasX, a C2c1, a C2c3, an engineered RNA-guided nuclease,and a codon-optimized RNA-guided nuclease; in an example, the cell (e.g., a plant cell or plant protoplast) stably or transiently expressesthe RNA-guided nuclease. In certain embodiments, the polynucleotide thatencodes the RNA-guided nuclease is, for example, DNA that encodes theRNA-guided nuclease and is stably integrated in the genome of a plantcell or plant protoplast, DNA or RNA that encodes the RNA-guidednuclease and is transiently present in or introduced into a plant cellor plant protoplast; such DNA or RNA can be introduced, e. g., by usinga vector such as a plasmid or viral vector or as an mRNA, or asvector-less DNA or RNA introduced directly into a plant cell or plantprotoplast.

In embodiments, the RNA-guided nuclease is provided as aribonucleoprotein (RNP) complex, e. g., a preassembled RNP that includesthe RNA-guided nuclease complexed with a polynucleotide including thegRNA or encoding a gRNA, or a preassembled RNP that includes apolynucleotide that encodes the RNA-guided nuclease (and optionallyencodes the gRNA, or is provided with a separate polynucleotideincluding the gRNA or encoding a gRNA), complexed with a protein. Incertain embodiments, the RNA-guided nuclease is a fusion protein, i. e.,wherein the RNA-guided nuclease (e. g., Cas9, Cpfl, CasY, CasX, C2c1, orC2c3) is covalently bound through a peptide bond to a cell-penetratingpeptide, a nuclear localization signal peptide, a chloroplast transitpeptide, or a mitochondrial targeting peptide; such fusion proteins areconveniently encoded in a single nucleotide sequence, optionallyincluding codons for linking amino acids. In certain embodiments, theRNA-guided nuclease or a polynucleotide that encodes the RNA-guidednuclease is provided as a complex with a cell-penetrating peptide orother transfecting agent. In certain embodiments, the RNA-guidednuclease or a polynucleotide that encodes the RNA-guided nuclease iscomplexed with, or covalently or non-covalently bound to, a furtherelement, e. g., a carrier molecule, an antibody, an antigen, a viralmovement protein, a polymer, a detectable label (e. g., a moietydetectable by fluorescence, radioactivity, or enzymatic orimmunochemical reaction), a quantum dot, or a particulate ornanoparticulate. In certain embodiments, the RNA-guided nuclease or apolynucleotide that encodes the RNA-guided nuclease is provided in asolution, or is provided in a liposome, micelle, emulsion, reverseemulsion, suspension, or other mixed-phase composition. In certainembodiments, the DNA or RNA is introduced directly into a plant cell orplant protoplast.

An RNA-guided nuclease can be provided to a cell (e. g., a plant cell orplant protoplast) by any suitable technique. In certain embodiments, theRNA-guided nuclease is provided by directly contacting a plant cell orplant protoplast with the RNA-guided nuclease or the polynucleotide thatencodes the RNA-guided nuclease. In certain embodiments, the RNA-guidednuclease is provided by transporting the RNA-guided nuclease or apolynucleotide that encodes the RNA-guided nuclease into a plant cell orplant protoplast using a chemical, enzymatic, or physical agent asprovided in detail in the paragraphs following the heading “DeliveryMethods and Delivery Agents”. In certain embodiments, the RNA-guidednuclease is provided by bacterially mediated (e. g., Agrobacterium sp.,Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp.,Azobacter sp., Phyllobacterium sp.) transfection of a plant cell orplant protoplast with a polynucleotide encoding the RNA-guided nuclease;see, e. g., Broothaerts et al. (2005) Nature, 433:629-633. In anembodiment, the RNA-guided nuclease is provided by transcription in aplant cell or plant protoplast of a DNA that encodes the RNA-guidednuclease and is stably integrated in the genome of the plant cell orplant protoplast or that is provided to the plant cell or plantprotoplast in the form of a plasmid or expression vector (e. g., a viralvector) that encodes the RNA-guided nuclease (and optionally encodes oneor more gRNAs, crRNAs, or sgRNAs, or is optionally provided with aseparate plasmid or vector that encodes one or more gRNAs, crRNAs, orsgRNAs). In certain embodiments, the RNA-guided nuclease is provided tothe plant cell or plant protoplast as a polynucleotide that encodes theRNA-guided nuclease, e. g., in the form of an mRNA encoding thenuclease.

Where a polynucleotide is concerned (e. g., a crRNA that includes thegRNA together with a separate tracrRNA, or a crRNA and a tracrRNAencoded on a single polynucleotide or on separate polynucleotides, or atleast one polynucleotide that is processed into one or more crRNAs and atracrRNA, or a sgRNA that includes the gRNA, or a polynucleotide thatencodes a sgRNA, or a polynucleotide that is processed into a sgRNA, ora polynucleotide that encodes the RNA-guided nuclease), embodiments ofthe polynucleotide include: (a) double-stranded RNA; (b) single-strandedRNA; (c) chemically modified RNA; (d) double-stranded DNA; (e)single-stranded DNA; (f) chemically modified DNA; or (g) a combinationof (a)-(f). Where expression of a polynucleotide is involved (e. g.,expression of a crRNA from a DNA encoding the crRNA, or expression andtranslation of an RNA-guided nuclease from a DNA encoding the nuclease),in some embodiments it is sufficient that expression be transient, i.e., not necessarily permanent or stable in the cell. Certain embodimentsof the polynucleotide further include additional nucleotide sequencesthat provide useful functionality; non-limiting examples of suchadditional nucleotide sequences include an aptamer or riboswitchsequence, nucleotide sequence that provides secondary structure such asstem-loops or that provides a sequence-specific site for an enzyme (e.g., a sequence-specific recombinase or endonuclease site), T-DNA (e. g.,DNA sequence encoding a gRNA, crRNA, tracrRNA, or sgRNA is enclosedbetween left and right T-DNA borders from Agrobacterium spp. or fromother bacteria that infect or induce tumours in plants), a DNAnuclear-targeting sequence, a regulatory sequence such as a promotersequence, and a transcript-stabilizing sequence. Certain embodiments ofthe polynucleotide include those wherein the polynucleotide is complexedwith, or covalently or non-covalently bound to, a non-nucleic acidelement, e. g., a carrier molecule, an antibody, an antigen, a viralmovement protein, a cell-penetrating or pore-forming peptide, a polymer,a detectable label, a quantum dot, or a particulate or nanoparticulate.

Apart from the CRISPR-type nucleases, other nucleases capable ofeffecting site-specific alteration or modification of a targetnucleotide sequence in the plant cell cultures, systems, methods, andcompositions provided herein include zinc-finger nucleases (ZFNs),transcription activator-like effector nucleases (TAL-effector nucleasesor TALENs), Argonaute proteins, and a meganuclease or engineeredmeganuclease. Zinc finger nucleases (ZFNs) are engineered proteinscomprising a zinc finger DNA-binding domain fused to a nucleic acidcleavage domain, e. g., a nuclease. The zinc finger binding domainsprovide specificity and can be engineered to specifically recognize anydesired target DNA sequence. For a review of the construction and use ofZFNs in plants and other organisms, see, e. g., Urnov et al. (2010)Nature Rev. Genet., 11:636-646. The zinc finger DNA binding domains arederived from the DNA-binding domain of a large class of eukaryotictranscription factors called zinc finger proteins (ZFPs). TheDNA-binding domain of ZFPs typically contains a tandem array of at leastthree zinc “fingers” each recognizing a specific triplet of DNA. Anumber of strategies can be used to design the binding specificity ofthe zinc finger binding domain. One approach, termed “modular assembly”,relies on the functional autonomy of individual zinc fingers with DNA.In this approach, a given sequence is targeted by identifying zincfingers for each component triplet in the sequence and linking them intoa multifinger peptide. Several alternative strategies for designing zincfinger DNA binding domains have also been developed. These methods aredesigned to accommodate the ability of zinc fingers to contactneighboring fingers as well as nucleotides bases outside their targettriplet. Typically, the engineered zinc finger DNA binding domain has anovel binding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, for example, rational design andvarious types of selection. Rational design includes, for example, theuse of databases of triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, e. g., U.S. Pat. Nos. 6,453,242 and 6,534,261,both incorporated herein by reference in their entirety. Exemplaryselection methods (e. g., phage display and yeast two-hybrid systems)are well known and described in the literature. In addition, enhancementof binding specificity for zinc finger binding domains has beendescribed in U.S. Pat. No. 6,794,136, incorporated herein by referencein its entirety. In addition, individual zinc finger domains may belinked together using any suitable linker sequences. Examples of linkersequences are publicly known, e. g., see U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949, incorporated herein by reference in theirentirety. The nucleic acid cleavage domain is non-specific and istypically a restriction endonuclease, such as Fokl. This endonucleasemust dimerize to cleave DNA. Thus, cleavage by Fokl as part of a ZFNrequires two adjacent and independent binding events, which must occurin both the correct orientation and with appropriate spacing to permitdimer formation. The requirement for two DNA binding events enables morespecific targeting of long and potentially unique recognition sites.Fokl variants with enhanced activities have been described; see, e.g.,Guo et al. (2010) J. Mol. Biol., 400:96-107.

Transcription activator like effectors (TALEs) are proteins secreted bycertain Xanthomonas species to modulate gene expression in host plantsand to facilitate the colonization by and survival of the bacterium.TALEs act as transcription factors and modulate expression of resistancegenes in the plants. Recent studies of TALEs have revealed the codelinking the repetitive region of TALEs with their target DNA-bindingsites. TALEs comprise a highly conserved and repetitive regionconsisting of tandem repeats of mostly 33 or 34 amino acid segments. Therepeat monomers differ from each other mainly at amino acid positions 12and 13. A strong correlation between unique pairs of amino acids atpositions 12 and 13 and the corresponding nucleotide in the TALE-bindingsite has been found. The simple relationship between amino acid sequenceand DNA recognition of the TALE binding domain allows for the design ofDNA binding domains of any desired specificity. TALEs can be linked to anon-specific DNA cleavage domain to prepare genome editing proteins,referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs,a restriction endonuclease, such as Fokl, can be conveniently used. Fora description of the use of TALENs in plants, see Mahfouz et al. (2011)Proc. Natl. Acad. Sci. USA, 108:2623-2628 and Mahfouz (2011) GM Crops,2:99-103.

Argonautes are proteins that can function as sequence-specificendonucleases by binding a polynucleotide (e. g., a single-stranded DNAor single-stranded RNA) that includes sequence complementary to a targetnucleotide sequence) that guides the Argonaut to the target nucleotidesequence and effects site-specific alteration of the target nucleotidesequence; see, e. g., US Patent Application Publication 2015/0089681,incorporated herein by reference in its entirety.

In related embodiments, zinc finger nucleases, TALENs, and Argonautesare used in conjunction with other functional domains. For example, thenuclease activity of these nucleic acid targeting systems can be alteredso that the enzyme binds to but does not cleave the DNA. Examples offunctional domains include transposase domains, integrase domains,recombinase domains, resolvase domains, invertase domains, proteasedomains, DNA methyltransferase domains, DNA hydroxylmethylase domains,DNA demethylase domains, histone acetylase domains, histone deacetylasedomains, nuclease domains, repressor domains, activator domains,nuclear-localization signal domains, transcription-regulatory protein(or transcription complex recruiting) domains, cellular uptake activityassociated domains, nucleic acid binding domains, antibody presentationdomains, histone modifying enzymes, recruiter of histone modifyingenzymes; inhibitor of histone modifying enzymes, histonemethyltransferases, histone demethylases, histone kinases, histonephosphatases, histone ribosylases, histone deribosylases, histoneubiquitinases, histone deubiquitinases, histone biotinases and histonetail proteases. Non-limiting examples of functional domains include atranscriptional activation domain, a transcription repression domain,and an SHH1, SUVH2, or SUVH9 polypeptide capable of reducing expressionof a target nucleotide sequence via epigenetic modification; see, e. g.,US Patent Application Publication 2016/0017348, incorporated herein byreference in its entirety. Genomic DNA may also be modified via baseediting using a fusion between a catalytically inactive Cas9 (dCas9) isfused to a cytidine deaminase which convert cytosine (C) to uridine (U),thereby effecting a C to T substitution; see Komor et al. (2016) Nature,533:420-424.

In some embodiments, one or more vectors driving expression of one ormore polynucleotides encoding elements of a genome-editing system (e.g., encoding a guide RNA or a nuclease) are introduced into a plant cellor a plant protoplast, whereby these elements, when expressed, result inalteration of a target nucleotide sequence. In certain embodiments, avector comprises a regulatory element such as a promoter operably linkedto one or more polynucleotides encoding elements of a genome-editingsystem. In such embodiments, expression of these polynucleotides can becontrolled by selection of the appropriate promoter, particularlypromoters functional in a plant cell; useful promoters includeconstitutive, conditional, inducible, and temporally or spatiallyspecific promoters (e. g., a tissue specific promoter, a developmentallyregulated promoter, or a cell cycle regulated promoter). In certainembodiments, the promoter is operably linked to nucleotide sequencesencoding multiple guide RNAs, wherein the sequences encoding guide RNAsare separated by a cleavage site such as a nucleotide sequence encodinga microRNA recognition/cleavage site or a self-cleaving ribozyme (see,e. g., Ferré-D'Amaré and Scott (2014) Cold Spring Harbor PerspectivesBiol., 2:a003574). In certain embodiments, the promoter is a pol IIpromoter operably linked to a nucleotide sequence encoding one or moreguide RNAs. In certain embodiments, the promoter operably linked to oneor more polynucleotides encoding elements of a genome-editing system isa constitutive promoter that drives DNA expression in plant cells; incertain embodiments, the promoter drives DNA expression in the nucleusor in an organelle such as a chloroplast or mitochondrion. Examples ofconstitutive promoters include a CaMV 35S promoter as disclosed in U.S.Pat. Nos. 5,858,742 and 5,322,938, a rice actin promoter as disclosed inU.S. Pat. No. 5,641,876, a maize chloroplast aldolase promoter asdisclosed in U.S. Pat. No. 7,151,204, and a opaline synthase (NOS) andoctapine synthase (OCS) promoter from Agrobacterium tumefaciens. Incertain embodiments, the promoter operably linked to one or morepolynucleotides encoding elements of a genome-editing system is apromoter from figwort mosaic virus (FMV), a RUBISCO promoter, or apyruvate phosphate dikinase (PDK) promoter, which is active in thechloroplasts of mesophyll cells. Other contemplated promoters includecell-specific or tissue-specific or developmentally regulated promoters,for example, a promoter that limits the expression of the nucleic acidtargeting system to germline or reproductive cells (e. g., promoters ofgenes encoding DNA ligases, recombinases, replicases, or other genesspecifically expressed in germline or reproductive cells); in suchembodiments, the nuclease-mediated genetic modification (e. g.,chromosomal or episomal double-stranded DNA cleavage) is limited onlythose cells from which DNA is inherited in subsequent generations, whichis advantageous where it is desirable that expression of thegenome-editing system be limited in order to avoid genotoxicity or otherunwanted effects. All of the patent publications referenced in thisparagraph are incorporated herein by reference in their entirety.

In some embodiments, elements of a genome-editing system (e. g., one ormore polynucleotides encoding an RNA-guided nuclease and a guide RNA andoptionally a donor template polynucleotide or one or morepolynucleotide(s) encoding a sequence-specific endonuclease and a donortemplate polynucleotide) are operably linked to separate regulatoryelements on separate vectors. In other embodiments, two or more elementsof a genome-editing system expressed from the same or differentregulatory elements or promoters are combined in a single vector,optionally with one or more additional vectors providing any additionalnecessary elements of a genome-editing system not included in the firstvector. For example, multiple guide RNAs can be expressed from onevector, with the appropriate RNA-guided nuclease expressed from a secondvector. In another example, one or more vectors for the expression ofone or more guide RNAs (e. g., crRNAs or sgRNAs) are delivered to a cell(e. g., a plant cell or a plant protoplast) that expresses theappropriate RNA-guided nuclease, or to a cell that otherwise containsthe nuclease, such as by way of prior administration thereto of a vectorfor in vivo expression of the nuclease.

Genome-editing system elements that are combined in a single vector maybe arranged in any suitable orientation, such as one element located 5′with respect to (“upstream” of) or 3′ with respect to (“downstream” of)a second element. The coding sequence of one element may be located onthe same or opposite strand of the coding sequence of a second element,and oriented in the same or opposite direction. In certain embodiments,the endonuclease and the nucleic acid-targeting guide RNA may beoperably linked to and expressed from the same promoter. In certainembodiments, a single promoter drives expression of a transcriptencoding an endonuclease and the guide RNA, embedded within one or moreintron sequences (e. g., each in a different intron, two or more in atleast one intron, or all in a single intron), which can beplant-derived; such use of introns is especially contemplated when theexpression vector is being transformed or transfected into a monocotplant cell or a monocot plant protoplast.

Expression vectors provided herein may contain a DNA segment near the 3′end of an expression cassette that acts as a signal to terminatetranscription and directs polyadenylation of the resultant mRNA. Such a3′ element is commonly referred to as a “3′-untranslated region” or“3′-UTR” or a “polyadenylation signal”. Useful 3′ elements include:Agrobacterium tumefaciens nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, andtr7 3′ elements disclosed in U.S. Pat. No. 6,090,627, incorporatedherein by reference, and 3′ elements from plant genes such as the heatshock protein 17, ubiquitin, and fructose-1,6-biphosphatase genes fromwheat (Triticum aestivum), and the glutelin, lactate dehydrogenase, andbeta-tubulin genes from rice (Oryza sativa), disclosed in US PatentApplication Publication 2002/0192813 A1, incorporated herein byreference.

In certain embodiments, a vector or an expression cassette includesadditional components, e. g., a polynucleotide encoding a drugresistance or herbicide gene or a polynucleotide encoding a detectablemarker such as green fluorescent protein (GFP) or beta-glucuronidase(gus) to allow convenient screening or selection of cells expressing thevector. In certain embodiments, the vector or expression cassetteincludes additional elements for improving delivery to a plant cell orplant protoplast or for directing or modifying expression of one or moregenome-editing system elements, for example, fusing a sequence encodinga cell-penetrating peptide, localization signal, transit, or targetingpeptide to the RNA-guided nuclease, or adding a nucleotide sequence tostabilize a guide RNA; such fusion proteins (and the polypeptidesencoding such fusion proteins) or combination polypeptides, as well asexpression cassettes and vectors for their expression in a cell, arespecifically claimed. In certain embodiments, an RNA-guided nuclease (e.g., Cas9, Cpfl, CasY, CasX, C2c1, or C2c3) is fused to a localizationsignal, transit, or targeting peptide, e. g., a nuclear localizationsignal (NLS), a chloroplast transit peptide (CTP), or a mitochondrialtargeting peptide (MTP); in a vector or an expression cassette, thenucleotide sequence encoding any of these can be located either 5′and/or 3′ to the DNA encoding the nuclease. For example, aplant-codon-optimized Cas9 (pco-Cas9) from Streptococcus pyogenes and S.thermophilus containing nuclear localization signals and codon-optimizedfor expression in maize is disclosed in PCT/US2015/018104 (published asWO/2015/131101 and claiming priority to U.S. Provisional PatentApplication 61/945,700), incorporated herein by reference. In anotherexample, a chloroplast-targeting RNA is appended to the 5′ end of anmRNA encoding an endonuclease to drive the accumulation of the mRNA inchloroplasts; see Gomez, et al. (2010) Plant Signal Behav., 5:1517-1519. In an embodiment, a Cas9 from Streptococcus pyogenes is fusedto a nuclear localization signal (NLS), such as the NLS from SV40. In anembodiment, a Cas9 from Streptococcus pyogenes is fused to acell-penetrating peptide (CPP), such as octa-arginine or nona-arginineor a homoarginine 12-mer oligopeptide, or a CPP disclosed in thedatabase of cell-penetrating peptides CPPsite 2.0, publicly available atcrdd[dot]osdd[dot]net/raghava/cppsite/. In an embodiment, a Cas9 fromStreptococcus pyogenes is fused to a chloroplast transit peptide (CTP)sequence. In certain embodiments, a CTP sequence is obtained from anynuclear gene that encodes a protein that targets a chloroplast, and theisolated or synthesized CTP DNA is appended to the 5′ end of the DNAthat encodes a nuclease targeted for use in a chloroplast. Chloroplasttransit peptides and their use are described in U.S. Pat. Nos.5,188,642, 5,728,925, and 8,420,888, all of which are incorporatedherein by reference in their entirety. Specifically, the CTP nucleotidesequences provided with the sequence identifier (SEQ ID) numbers 12-15and 17-22 of U.S. Pat. No. 8,420,888 are incorporated herein byreference. In an embodiment, a Cas9 from Streptococcus pyogenes is fusedto a mitochondrial targeting peptide (MTP), such as a plant MTPsequence; see, e. g., Jores et al. (2016) Nature Communications,7:12036-12051.

Plasmids designed for use in plants and encoding CRISPR genome editingelements (CRISPR nucleases and guide RNAs) are publicly available fromplasmid repositories such as Addgene (Cambridge, Mass.; also see“addgene[dot]com”) or can be designed using publicly disclosedsequences, e. g., sequences of CRISPR nucleases. In certain embodiments,such plasmids are used to co-express both CRISPR nuclease mRNA and guideRNA(s); in other embodiments, CRISPR endonuclease mRNA and guide RNA areencoded on separate plasmids. In certain embodiments, the plasmids areAgrobacterium TI plasmids. Materials and methods for preparingexpression cassettes and vectors for CRISPR endonuclease and guide RNAfor stably integrated and/or transient plant transformation aredisclosed in PCT/US2015/018104 (published as WO/2015/131101 and claimingpriority to U.S. Provisional Patent Application 61/945,700), US PatentApplication Publication 2015/0082478 A1, and PCT/US2015/038767(published as WO/2016/007347 and claiming priority to U.S. ProvisionalPatent Application 62/023,246), all of which are incorporated herein byreference in their entirety. In certain embodiments, such expressioncassettes are isolated linear fragments, or are part of a largerconstruct that includes bacterial replication elements and selectablemarkers; such embodiments are useful, e. g., for particle bombardment ornanoparticle delivery or protoplast transformation. In certainembodiments, the expression cassette is adjacent to or located betweenT-DNA borders or contained within a binary vector, e. g., forAgrobacterium-mediated transformation. In certain embodiments, a plasmidencoding a CRISPR nuclease is delivered to cell (such as a plant cell ora plant protoplast) for stable integration of the CRISPR nuclease intothe genome of cell, or alternatively for transient expression of theCRISPR nuclease. In certain embodiments, plasmids encoding a CRISPRnuclease are delivered to a plant cell or a plant protoplast to achievestable or transient expression of the CRISPR nuclease, and one ormultiple guide RNAs (such as a library of individual guide RNAs ormultiple pooled guide RNAs) or plasmids encoding the guide RNAs aredelivered to the plant cell or plant protoplast individually or incombinations, thus providing libraries or arrays of plant cells or plantprotoplasts (or of plant callus or whole plants derived therefrom), inwhich a variety of genome edits are provided by the different guideRNAs.

In certain embodiments where the genome-editing system is a CRISPRsystem, expression of the guide RNA is driven by a plant U6 spliceosomalRNA promoter, which can be native to the genome of the plant cell orfrom a different species, e. g., a U6 promoter from maize, tomato, orsoybean such as those disclosed in PCT/US2015/018104 (published as WO2015/131101 and claiming priority to U.S. Provisional Patent Application61/945,700), incorporated herein by reference, or a homologue thereof;such a promoter is operably linked to DNA encoding the guide RNA fordirecting an endonuclease, followed by a suitable 3′ element such as aU6 poly-T terminator. In another embodiment, an expression cassette forexpressing guide RNAs in plants is used, wherein the promoter is a plantU3, 7SL (signal recognition particle RNA), U2, or U5 promoter, orchimerics thereof, e. g., as described in PCT/US2015/018104 (publishedas WO 2015/131101 and claiming priority to U.S. Provisional PatentApplication 61/945,700), incorporated herein by reference. When multipleor different guide RNA sequences are used, a single expression constructmay be used to correspondingly direct the genome editing activity to themultiple or different target sequences in a cell, such a plant cell or aplant protoplast. In various embodiments, a single vector includes 1, 2,3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, or more guide RNAsequences; in other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about15, about 20, or more guide RNA sequences are provided on multiplevectors, which can be delivered to one or multiple plant cells or plantprotoplasts (e. g., delivered to an array of plant cells or plantprotoplasts, or to a pooled population of plant cells or plantprotoplasts).

In embodiments, one or more guide RNAs and the corresponding RNA-guidednuclease are delivered together or simultaneously. In other embodiments,one or more guide RNAs and the corresponding RNA-guided nuclease aredelivered separately; these can be delivered in separate, discrete stepsand using the same or different delivery techniques. In an example, anRNA-guided nuclease is delivered to a cell (such as a plant cell orplant protoplast) by particle bombardment, on carbon nanotubes, or byAgrobacterium-mediated transformation, and one or more guide RNAs isdelivered to the cell in a separate step using the same or differentdelivery technique. In certain embodiments, an RNA-guided nucleaseencoded by a DNA molecule or an mRNA is delivered to a cell with enoughtime prior to delivery of the guide RNA to permit expression of thenuclease in the cell; for example, an RNA-guided nuclease encoded by aDNA molecule or an mRNA is delivered to a plant cell or plant protoplastbetween or about 1-12 hours (e. g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 hours, or between about or about 1-6 hours or between about orabout 2-6 hours) prior to the delivery of the guide RNA to the plantcell or plant protoplast. In certain embodiments, whether the RNA-guidednuclease is delivered simultaneously with or separately from an initialdose of guide RNA, succeeding “booster” doses of guide RNA are deliveredsubsequent to the delivery of the initial dose; for example, a second“booster” dose of guide RNA is delivered to a plant cell or plantprotoplast between about or about 1-12 hours (e. g., about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 hours, or between about or about 1-6 hoursor between about or about 2-6 hours) subsequent to the delivery of theinitial dose of guide RNA to the plant cell or plant protoplast.Similarly, in some embodiments, multiple deliveries of an RNA-guidednuclease or of a DNA molecule or an mRNA encoding an RNA-guided nucleaseare used to increase efficiency of the genome modification.

In embodiments, the desired genome modification involves homologousrecombination, wherein one or more double-stranded DNA break in thetarget nucleotide sequence is generated by the RNA-guided nuclease andguide RNA(s), followed by repair of the break(s) using a homologousrecombination mechanism (“homology-directed repair”). In suchembodiments, a donor template that encodes the desired nucleotidesequence to be inserted or knocked-in at the double-stranded break isprovided to the cell (such as a plant cell or plant protoplast);examples of suitable templates include single-stranded DNA templates anddouble-stranded DNA templates (e. g., in the form of a plasmid). Ingeneral, a donor template encoding a nucleotide change over a region ofless than about 50 nucleotides is conveniently provided in the form ofsingle-stranded DNA; larger donor templates (e. g., more than 100nucleotides) are often conveniently provided as double-stranded DNAplasmids. In certain embodiments, the various compositions and methodsdescribed herein for delivering guide RNAs and nucleases are alsogenerally useful for delivering the donor template polynucleotide to thecell; this delivery can be simultaneous with, or separate from(generally after) delivery of the nuclease and guide RNA to the cell.For example, a donor template can be transiently introduced into a plantcell or plant protoplast, optionally with the nuclease and/or gRNA; incertain embodiments, the donor template is provided to the plant cell orplant protoplast in a quantity that is sufficient to achieve the desiredhomology-directed repair but that does not persist in the plant cell orplant protoplast after a given period of time (e. g., after one or morecell division cycles). In certain embodiments, a donor template has acore nucleotide sequence that differs from the target nucleotidesequence (e. g., a homologous endogenous genomic region) by at least 1,at least 5, at least 10, at least 20, at least 30, at least 40, at least50, or more nucleotides. This core sequence is flanked by “homologyarms” or regions of high sequence identity with the targeted nucleotidesequence; in certain embodiments, the regions of high identity includeat least 10, at least 50, at least 100, at least 150, at least 200, atleast 300, at least 400, at least 500, at least 600, at least 750, or atleast 1000 nucleotides on each side of the core sequence. In certainembodiments where the donor template is in the form of a single-strandedDNA, the core sequence is flanked by homology arms including at least10, at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, or at least 100 nucleotides on each side of thecore sequence. In certain embodiments where the donor template is in theform of a double-stranded DNA plasmid, the core sequence is flanked byhomology arms including at least 500, at least 600, at least 700, atleast 800, at least 900, or at least 1000 nucleotides on each side ofthe core sequence. In an embodiment, two separate double-strand breaksare introduced into the cell's target nucleotide sequence with a “doublenickase” Cas9 (see Ran et al. (2013) Cell, 154:1380-1389), followed bydelivery of the donor template.

Delivery Methods and Delivery Agents: Various treatments are useful indelivery of a polynucleotide, including a guide RNA (gRNA), such as acrRNA or sgRNA (or a polynucleotide encoding such), donor templatepolynucleotide, polynucleotide encoding a nuclease (e.g., RNA-guidednuclease or sequence-specific endonuclease), or a polynucleotideencoding an ROS scavenging agent to a plant cell or plant protoplast. Incertain embodiments, one or more treatments is employed to deliver thegRNA or other polynucleotide (e.g., donor template polynucleotide, or apolynucleotide encoding an ROS scavenging agent) into a plant, plantcell or plant protoplast, e. g., through barriers such as a cell wall ora plasma membrane or nuclear envelope or other lipid bilayer. In anembodiment, a gRNA- or other polynucleotide-containing composition isdelivered directly, for example by direct contact of the polynucleotidecomposition with a plant cell or plant protoplast. A gRNA- or otherpolynucleotide (e.g., donor template polynucleotide, polynucleotideencoding a nuclease, or a polynucleotide encoding an ROS scavengingagent)-containing composition in the form of a liquid, a solution, asuspension, an emulsion, a reverse emulsion, a colloid, a dispersion, agel, liposomes, micelles, an injectable material, an aerosol, a solid, apowder, a particulate, a nanoparticle, or a combination thereof can beapplied directly to a plant cell or plant protoplast (e. g., throughabrasion or puncture or otherwise disruption of the cell wall or cellmembrane, by spraying or dipping or soaking or otherwise directlycontacting, by microinjection). For example, a plant cell or plantprotoplast is soaked in a liquid gRNA- or otherpolynucleotide-containing composition, whereby the gRNA or otherpolynucleotide is delivered to the plant cell or plant protoplast. Incertain embodiments, the gRNA- or other polynucleotide-containingcomposition is delivered using negative or positive pressure, forexample, using vacuum infiltration or application of hydrodynamic orfluid pressure. In certain embodiments, the gRNA- or otherpolynucleotide-containing composition is introduced into a plant cell orplant protoplast, e. g., by microinjection or by disruption ordeformation of the cell wall or cell membrane, for example by physicaltreatments such as by application of negative or positive pressure,shear forces, or treatment with a chemical or physical delivery agentsuch as surfactants, liposomes, or nanoparticles; see, e. g., deliveryof materials to cells employing microfluidic flow through acell-deforming constriction as described in US Published PatentApplication 2014/0287509, incorporated by reference in its entiretyherein. Other techniques useful for delivering the gRNA- or otherpolynucleotide-containing composition to a plant cell or plantprotoplast include: ultrasound or sonication; vibration, friction, shearstress, vortexing, cavitation; centrifugation or application ofmechanical force; mechanical cell wall or cell membrane deformation orbreakage; enzymatic cell wall or cell membrane breakage orpermeabilization; abrasion or mechanical scarification (e. g., abrasionwith carborundum or other particulate abrasive or scarification with afile or sandpaper) or chemical scarification (e. g., treatment with anacid or caustic agent); and electroporation. In certain embodiments, thegRNA- or other polynucleotide-containing composition is provided bybacterially mediated (e. g., Agrobacterium sp., Rhizobium sp.,Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp.,Phyllobacterium sp.) transfection of the plant cell or plant protoplastwith a polynucleotide encoding the gRNA; see, e. g., Broothaerts et al.(2005) Nature, 433:629-633. Any of these techniques or a combinationthereof are alternatively employed on the plant part or tissue or intactplant (or seed) from which a plant cell or plant protoplast isoptionally subsequently obtained or isolated; in certain embodiments,the gRNA- or other polynucleotide (e.g., donor template polynucleotide,polynucleotide encoding a nuclease, or a polynucleotide encoding an ROSscavenging agent)-containing composition is delivered in a separate stepafter the plant cell or plant protoplast has been obtained or isolated.

In embodiments, a treatment employed in delivery of a polynucleotideincluding gRNA, a donor template polynucleotide, or a polynucleotideencoding an ROS scavenging agent to a plant cell or plant protoplast iscarried out under a specific thermal regime, which can involve one ormore appropriate temperatures, e. g., chilling or cold stress (exposureto temperatures below that at which normal plant growth occurs), orheating or heat stress (exposure to temperatures above that at whichnormal plant growth occurs), or treating at a combination of differenttemperatures. In certain embodiments, a specific thermal regime iscarried out on the plant cell or plant protoplast, or on a plant orplant part from which a plant cell or plant protoplast is subsequentlyobtained or isolated, in one or more steps separate from the gRNA, thedonor template polynucleotide, or the ROS scavenging agentpolynucleotide delivery.

In embodiments of the cultures, systems, methods, and compositionsprovided herein, a whole plant or plant part or seed, or an isolatedplant cell or plant protoplast, or the plant or plant part from which aplant cell or plant protoplast is obtained or isolated, is treated withone or more delivery agents which can include at least one chemical,enzymatic, or physical agent, or a combination thereof. In certainembodiments, polynucleotide, e.g., a gRNA-, a donor templatepolynucleotide-, or a polynucleotide encoding an ROS scavenging agentcontaining composition further includes one or more one chemical,enzymatic, or physical agents for delivery. In certain embodiments thatfurther include the step of providing an RNA-guided nuclease or apolynucleotide that encodes the RNA-guided nuclease, a gRNA-containingcomposition including the RNA-guided nuclease or polynucleotide thatencodes the RNA-guided nuclease further includes one or more onechemical, enzymatic, or physical agent for delivery. In certainembodiments that further include the step of providing asequence-specific endonuclease or a polynucleotide that encodes thesequence-specific endonuclease, a donor templatepolynucleotide-containing composition including the sequence specificendonuclease or polynucleotide that encodes the sequence specificendonuclease can further comprise one or more one chemical, enzymatic,or physical agents for delivery. Treatment with the chemical, enzymaticor physical agent can be carried out simultaneously with the gRNAdelivery, with the RNA-guided nuclease delivery, or in one or moreseparate steps that precede or follow the gRNA delivery or theRNA-guided nuclease delivery. In certain embodiments, a chemical,enzymatic, or physical agent, or a combination of these, is associatedor complexed with the polynucleotide composition, with the donortemplate polynucleotide, with the sequence specific endonuclease orpolynucleotide that encodes the sequence specific endonuclease, with thepolynucleotide encoding an ROS scavenging agent, with the gRNA orpolynucleotide that encodes or is processed to the gRNA, or with theRNA-guided nuclease or polynucleotide that encodes the RNA-guidednuclease; examples of such associations or complexes include thoseinvolving non-covalent interactions (e. g., ionic or electrostaticinteractions, hydrophobic or hydrophilic interactions, formation ofliposomes, micelles, or other heterogeneous composition) and covalentinteractions (e. g., peptide bonds, bonds formed using cross-linkingagents). In non-limiting examples, a donor template polynucleotide, gRNAor polynucleotide that encodes or is processed to the gRNA is providedas a liposomal complex with a cationic lipid; a gRNA or polynucleotidethat encodes or is processed to the gRNA is provided as a complex with acarbon nanotube; and an RNA-guided nuclease is provided as a fusionprotein between the nuclease and a cell-penetrating peptide. Examples ofagents useful for delivering a donor template polynucleotide, gRNA orpolynucleotide that encodes or is processed to the gRNA or a nuclease orpolynucleotide that encodes the nuclease include the various cationicliposomes and polymer nanoparticles reviewed by Zhang et al. (2007) J.Controlled Release, 123:1-10, and the cross-linked multilamellarliposomes described in US Patent Application Publication 2014/0356414 A1, incorporated by reference in its entirety herein. In any of theaforementioned embodiments, it is further contemplated that otherpolynucleotides or polypeptides of interest including: (i) a donortemplate polynucleotide; (ii) a sequence specific endonuclease orpolynucleotide encoding a sequence specific endonuclease; (iii) acombination of (i) and (ii); or (iv) a polynucleotide encoding an ROSscavenging agent can be substituted for the aforementioned gRNA and/orRNA-guided nuclease or polynucleotide encoding the RNA-guided nuclease.

In certain embodiments, the chemical agent can comprise or is at leastone selected from the group consisting of:

(a) solvents (e. g., water, dimethylsulfoxide, dimethylformamide,acetonitrile, N-pyrrolidine, pyridine, hexamethylphosphoramide,alcohols, alkanes, alkenes, dioxanes, polyethylene glycol, and othersolvents miscible or emulsifiable with water or that will dissolvephosphonucleotides in non-aqueous systems);

(b) fluorocarbons (e. g., perfluorodecalin, perfluoromethyldecalin);

(c) glycols or polyols (e. g., propylene glycol, polyethylene glycol);

(d) surfactants, including cationic surfactants, anionic surfactants,non-ionic surfactants, and amphiphilic surfactants, e. g., alkyl or arylsulfates, phosphates, sulfonates, or carboxylates; primary, secondary,or tertiary amines; quaternary ammonium salts; sultaines, betaines;cationic lipids; phospholipids; tallowamine; bile acids such as cholicacid; long chain alcohols; organosilicone surfactants including nonionicorganosilicone surfactants such as trisiloxane ethoxylate surfactants ora silicone polyether copolymer such as a copolymer of polyalkylene oxidemodified heptamethyl trisiloxane and allyloxypolypropylene glycolmethylether (commercially available as SILWET L-77™ brand surfactanthaving CAS Number 27306-78-1 and EPA Number CAL. REG. NO. 5905-50073-AA,Momentive Performance Materials, Inc., Albany, N.Y.); specific examplesof useful surfactants include sodium lauryl sulfate, the Tween series ofsurfactants, Triton-X100, Triton-X114, CHAPS and CHAPSO, Tergitol-typeNP-40, Nonidet P-40;

(e) lipids, lipoproteins, lipopolysaccharides;

(f) acids, bases, caustic agents;

(g) peptides, proteins, or enzymes (e. g., cellulase, pectolyase,maceroenzyme, pectinase), including cell-penetrating or pore-formingpeptides (e. g., (BO100)2K8, Genscript; poly-lysine, poly-arginine, orpoly-homoarginine peptides; gamma zein, see US Patent Applicationpublication 2011/0247100, incorporated herein by reference in itsentirety; transcription activator of human immunodeficiency virus type 1(“HIV-1 Tat”) and other Tat proteins, see, e. g.,www[dot]lifetein[dot]com/Cell_Penetrating_Peptides[dot]html and Järver(2012) Mol. Therapy—Nucleic Acids, 1:e27, 1-17); octa-arginine ornona-arginine; poly-homoarginine (see Unnamalai et al. (2004) FEBSLetters, 566:307-310); see also the database of cell-penetratingpeptides CPPsite 2.0 publicly available atcrdd[dot]osdd[dot]net/raghava/cppsite/

(h) RNase inhibitors;

(i) cationic branched or linear polymers such as chitosan, poly-lysine,DEAE-dextran, polyvinylpyrrolidone (“PVP”), or polyethylenimine (“PEI”,e. g., PEI, branched, MW 25,000, CAS #9002-98-6; PEI, linear, MW 5000,CAS #9002-98-6; PEI linear, MW 2500, CAS #9002-98-6);

(j) dendrimers (see, e. g., US Patent Application Publication2011/0093982, incorporated herein by reference in its entirety);

(k) counter-ions, amines or polyamines (e. g., spermine, spermidine,putrescine), osmolytes, buffers, and salts (e. g., calcium phosphate,ammonium phosphate);

(l) polynucleotides (e. g., non-specific double-stranded DNA, salmonsperm DNA);

(m) transfection agents (e. g., Lipofectin®, Lipofectamine®, andOligofectamine®, and Invivofectamine® (all from Thermo FisherScientific, Waltham, Mass.), PepFect (see Ezzat et al. (2011) NucleicAcids Res., 39:5284-5298), TransIt® transfection reagents (Mirus Bio,LLC, Madison, Wis.), and poly-lysine, poly-homoarginine, andpoly-arginine molecules including octo-arginine and nono-arginine asdescribed in Lu et al. (2010) J. Agric. Food Chem., 58:2288-2294);

(n) antibiotics, including non-specific DNA double-strand-break-inducingagents (e. g., phleomycin, bleomycin, talisomycin); and/or

(o) antioxidants (e. g., glutathione, dithiothreitol, ascorbate).

In embodiments, the chemical agent is provided simultaneously with thepolynucleotide (e.g., donor template polynucleotide, gRNA (orpolynucleotide encoding the gRNA or that is processed to the gRNA), forexample, the polynucleotide composition including the gRNA furtherincludes one or more chemical agent. In certain embodiments, the gRNA orpolynucleotide encoding the gRNA or that is processed to the gRNA iscovalently or non-covalently linked or complexed with one or morechemical agent; for example, the gRNA or polynucleotide encoding thegRNA or that is processed to the gRNA can be covalently linked to apeptide or protein (e. g., a cell-penetrating peptide or a pore-formingpeptide) or non-covalently complexed with cationic lipids, polycations(e. g., polyamines), or cationic polymers (e. g., PEI). In certainembodiments, the gRNA or polynucleotide encoding the gRNA or that isprocessed to the gRNA is complexed with one or more chemical agents toform, e. g., a solution, liposome, micelle, emulsion, reverse emulsion,suspension, colloid, or gel. In any of the aforementioned embodiments,it is further contemplated that other polynucleotides or polypeptides ofinterest including: (i) a donor template polynucleotide; (ii) a sequencespecific endonuclease or polynucleotide encoding a sequence specificendonuclease; (iii) a combination of (i) and (ii); or (iv) apolynucleotide encoding an ROS scavenging agent can be substituted forthe aforementioned gRNA and/or RNA-guided nuclease or polynucleotideencoding the RNA-guided nuclease.

In embodiments, the physical agent (e.g., for delivery of apolynucleotide and/or polypeptide) is at least one selected from thegroup consisting of particles or nanoparticles (e. g., particles ornanoparticles made of materials such as carbon, silicon, siliconcarbide, gold, tungsten, polymers, or ceramics) in various size rangesand shapes, magnetic particles or nanoparticles (e. g., silenceMagMagnetotransfection™ agent, OZ Biosciences, San Diego, Calif.), abrasiveor scarifying agents, needles or microneedles, matrices, and grids. Incertain embodiments, particulates and nanoparticulates are useful indelivery of the polynucleotide composition or the nuclease or both.Useful particulates and nanoparticles include those made of metals (e.g., gold, silver, tungsten, iron, cerium), ceramics (e. g., aluminumoxide, silicon carbide, silicon nitride, tungsten carbide), polymers (e.g., polystyrene, polydiacetylene, and poly(3,4-ethylenedioxythiophene)hydrate), semiconductors (e. g., quantum dots), silicon (e. g., siliconcarbide), carbon (e. g., graphite, graphene, graphene oxide, or carbonnanosheets, nanocomplexes, or nanotubes), and composites (e. g.,polyvinylcarbazole/graphene, polystyrene/graphene, platinum/graphene,palladium/graphene nanocomposites). In certain embodiments, suchparticulates and nanoparticulates are further covalently ornon-covalently functionalized, or further include modifiers orcross-linked materials such as polymers (e. g., linear or branchedpolyethylenimine, poly-lysine), polynucleotides (e. g., DNA or RNA),polysaccharides, lipids, polyglycols (e. g., polyethylene glycol,thiolated polyethylene glycol), polypeptides or proteins, and detectablelabels (e. g., a fluorophore, an antigen, an antibody, or a quantumdot). In various embodiments, such particulates and nanoparticles areneutral, or carry a positive charge, or carry a negative charge.Embodiments of compositions including particulates include thoseformulated, e. g., as liquids, colloids, dispersions, suspensions,aerosols, gels, and solids. Embodiments include nanoparticles affixed toa surface or support, e. g., an array of carbon nanotubes verticallyaligned on a silicon or copper wafer substrate. Embodiments includepolynucleotide compositions including particulates (e. g., gold ortungsten or magnetic particles) delivered by a Biolistic-type techniqueor with magnetic force. The size of the particles used in Biolistics isgenerally in the “microparticle” range, for example, gold microcarriersin the 0.6, 1.0, and 1.6 micrometer size ranges (see, e. g., instructionmanual for the Helios® Gene Gun System, Bio-Rad, Hercules, Calif.;Randolph-Anderson et al. (2015) “Sub-micron gold particles are superiorto larger particles for efficient Biolistic® transformation oforganelles and some cell types”, Bio-Rad US/EG Bulletin 2015), butsuccessful Biolistics delivery using larger (40 nanometer) nanoparticleshas been reported in cultured animal cells; see O'Brian and Lummis(2011) BMC Biotechnol., 11:66-71. Other embodiments of usefulparticulates are nanoparticles, which are generally in the nanometer(nm) size range or less than 1 micrometer, e. g., with a diameter ofless than about 1 nm, less than about 3 nm, less than about 5 nm, lessthan about 10 nm, less than about 20 nm, less than about 40 nm, lessthan about 60 nm, less than about 80 nm, and less than about 100 nm.Specific, non-limiting embodiments of nanoparticles commerciallyavailable (all from Sigma-Aldrich Corp., St. Louis, Mo.) include goldnanoparticles with diameters of 5, 10, or 15 nm; silver nanoparticleswith particle sizes of 10, 20, 40, 60, or 100 nm; palladium “nanopowder”of less than 25 nm particle size; single-, double-, and multi-walledcarbon nanotubes, e. g., with diameters of 0.7-1.1, 1.3-2.3, 0.7-0.9, or0.7-1.3 nm, or with nanotube bundle dimensions of 2-10 nm by 1-5micrometers, 6-9 nm by 5 micrometers, 7-15 nm by 0.5-10 micrometers,7-12 nm by 0.5-10 micrometers, 110-170 nm by 5-9 micrometers, 6-13 nm by2.5-20 micrometers. Embodiments include polynucleotide compositionsincluding materials such as gold, silicon, cerium, or carbon, e. g.,gold or gold-coated nanoparticles, silicon carbide whiskers,carborundum, porous silica nanoparticles, gelatin/silica nanoparticles,nanoceria or cerium oxide nanoparticles (CNPs), carbon nanotubes (CNTs)such as single-, double-, or multi-walled carbon nanotubes and theirchemically functionalized versions (e. g., carbon nanotubesfunctionalized with amide, amino, carboxylic acid, sulfonic acid, orpolyethylene glycol moeities), and graphene or graphene oxide orgraphene complexes; see, for example, Wong et al. (2016) Nano Lett.,16:1161-1172; Giraldo et al. (2014) Nature Materials, 13:400-409; Shenet al. (2012) Theranostics, 2:283-294; Kim et al. (2011) BioconjugateChem., 22:2558-2567; Wang et al. (2010) J Am. Chem. Soc. Comm.,132:9274-9276; Zhao et al. (2016) Nanoscale Res. Lett., 11:195-203; andChoi et al. (2016) J. Controlled Release, 235:222-235. See also, forexample, the various types of particles and nanoparticles, theirpreparation, and methods for their use, e.g., in deliveringpolynucleotides and polypeptides to cells, disclosed in US PatentApplication Publications 2010/0311168, 2012/0023619, 2012/0244569,2013/0145488, 2013/0185823, 2014/0096284, 2015/0040268, 2015/0047074,and 2015/0208663, all of which are incorporated herein by reference intheir entirety.

In embodiments wherein the polynucleotide (e.g., gRNA (or polynucleotideencoding the gRNA) a donor template polynucleotide, polynucleotideencoding a sequence specific endonuclease, or a polynucleotide encodingan ROS scavenging agent) is provided in a composition that furtherincludes an RNA-guided nuclease (or a polynucleotide that encodes theRNA-guided nuclease), or wherein the method further includes the step ofproviding an RNA-guided nuclease or a polynucleotide that encodes theRNA-guided nuclease, one or more one chemical, enzymatic, or physicalagent can similarly be employed. In certain embodiments, the RNA-guidednuclease (or polynucleotide encoding the RNA-guided nuclease) isprovided separately, e. g., in a separate composition including theRNA-guided nuclease or polynucleotide encoding the RNA-guided nuclease.Such compositions can include other chemical or physical agents (e. g.,solvents, surfactants, proteins or enzymes, transfection agents,particulates or nanoparticulates), such as those described above asuseful in the polynucleotide composition used to provide the gRNA. Forexample, porous silica nanoparticles are useful for delivering a DNArecombinase into maize cells; see, e. g., Martin-Ortigosa et al. (2015)Plant Physiol., 164:537-547. In an embodiment, the polynucleotidecomposition includes a gRNA and Cas9 nuclease, and further includes asurfactant and a cell-penetrating peptide. In an embodiment, thepolynucleotide composition includes a plasmid that encodes both anRNA-guided nuclease and at least on gRNA, and further includes asurfactant and carbon nanotubes. In an embodiment, the polynucleotidecomposition includes multiple gRNAs and an mRNA encoding the RNA-guidednuclease, and further includes particles (e.g., gold or tungstenparticles), and the polynucleotide composition is delivered to a plantcell or plant protoplast by Biolistics. In any of the aforementionedembodiments, it is further contemplated that other polynucleotides orpolypeptides of interest including: (i) a donor template polynucleotide;(ii) a sequence specific endonuclease or polynucleotide encoding asequence specific endonuclease; (iii) a combination of (i) and (ii); or(iv) a polynucleotide encoding an ROS scavenging agent can besubstituted for the aforementioned gRNA and/or RNA-guided nuclease orpolynucleotide encoding the RNA-guided nuclease.

In related embodiments, one or more chemical, enzymatic, or physicalagent(s) can be used in one or more steps separate from (preceding orfollowing) that in which the polynucleotide (e.g., gRNA, a donortemplate polynucleotide, polynucleotide encoding a sequence specificendonuclease, or a polynucleotide encoding an ROS scavenging agent) isprovided. In an embodiment, the plant or plant part from which a plantcell or plant protoplast is obtained or isolated is treated with one ormore one chemical, enzymatic, or physical agent in the process ofobtaining or isolating the plant cell or plant protoplast. In certainembodiments, the plant or plant part is treated with an abrasive, acaustic agent, a surfactant such as Silwet L-77 or a cationic lipid, oran enzyme such as cellulase.

In embodiments, a polynucleotide, including a gRNA, a donor templatepolynucleotide, polynucleotide encoding a sequence specificendonuclease, or a polynucleotide encoding an ROS scavenging agent, isdelivered to plant cells or plant protoplasts prepared or obtained froma plant, plant part, or plant tissue that has been treated with thepolynucleotide compositions (and optionally the nuclease). In certainembodiments, one or more one chemical, enzymatic, or physical agent,separately or in combination with the polynucleotide composition, isprovided/applied at a location in the plant or plant part other than theplant location, part, or tissue from which the plant cell or plantprotoplast is obtained or isolated. In certain embodiments, thepolynucleotide composition is applied to adjacent or distal cells ortissues and is transported (e. g., through the vascular system or bycell-to-cell movement) to the meristem from which plant cells or plantprotoplasts are subsequently isolated. In certain embodiments, agRNA-containing composition is applied by soaking a seed or seedfragment or zygotic or somatic embryo in the gRNA-containingcomposition, whereby the gRNA is delivered to the seed or seed fragmentor zygotic or somatic embryo from which plant cells or plant protoplastsare subsequently isolated. In certain embodiments, a flower bud or shoottip is contacted with a gRNA-containing composition, whereby the gRNA isdelivered to cells in the flower bud or shoot tip from which plant cellsor plant protoplasts are subsequently isolated. In certain embodiments,a gRNA-containing composition is applied to the surface of a plant or ofa part of a plant (e. g., a leaf surface), whereby the gRNA is deliveredto tissues of the plant from which plant cells or plant protoplasts aresubsequently isolated. In certain embodiments a whole plant or planttissue is subjected to particle- or nanoparticle-mediated delivery (e.g., Biolistics or carbon nanotube or nanoparticle delivery) of agRNA-containing composition, whereby the gRNA is delivered to cells ortissues from which plant cells or plant protoplasts are subsequentlyisolated. In any of the aforementioned embodiments, it is furthercontemplated that other polynucleotides or polypeptides of interestincluding: (i) a donor template polynucleotide; (ii) a sequence specificendonuclease or polynucleotide encoding a sequence specificendonuclease; (iii) a combination of (i) and (ii); or (iv) apolynucleotide encoding an ROS scavenging agent can be substituted forthe aforementioned gRNA and/or RNA-guided nuclease or polynucleotideencoding the RNA-guided nuclease.

EMBODIMENTS

Various embodiments of the cultures, systems, methods, and compositionsprovided herein are included in the following non-limiting list ofembodiments.

Embodiment 1. A plant protoplast culture comprising:

-   -   (a) at least one plant protoplast; and    -   (b) a culture medium comprising: (i) at least 40 millimolar Ca²⁺        or Mg²⁺; (ii) an antioxidant; or (iii) a combination of (i) and        (ii).

Embodiment 2. The plant protoplast culture of embodiment 1, wherein theplant protoplast is obtained from a plant tissue, whole plant, intactnodal bud, shoot apex or shoot apical meristem, root apex or root apicalmeristem, lateral meristem, intercalary meristem, seedling, whole seed,halved seed or other seed fragment, zygotic embryo, somatic embryo,ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole,stem, tuber, root, callus, or plant cell suspension.

Embodiment 3. The plant protoplast culture of any one of embodiments 1to 2, wherein the plant protoplast is obtained from (a) a monocot plant,or (b) a dicot plant.

Embodiment 4. The plant protoplast culture of any one of embodiments 1to 3, wherein the plant protoplast is haploid, diploid, or polyploid.

Embodiment 5. The plant protoplast culture of any one of embodiments 1to 4, wherein the plant protoplast is obtained from alfalfa (Medicagosativa), almonds (Prunus dulcis), apples (Malus×domestica), apricots(Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P. sibirica),asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Hordeumvulgare), beans (Phaseolus spp.), blueberries and cranberries (Vacciniumspp.), cacao (Theobroma cacao), canola and rapeseed or oilseed rape,(Brassica napus), carnation (Dianthus caryophyllus), carrots (Daucuscarota sativus), cassava (Manihot esculentum), cherry (Prunus avium),chickpea (Cider arietinum), chicory (Cichorium intybus), chili peppersand other capsicum peppers (Capsicum annuum, C. frutescens, C. chinense,C. pubescens, C. baccatum), chrysanthemums (Chrysanthemum spp.), coconut(Cocos nucifera), coffee (Coffea spp. including Coffea arabica andCoffea canephora), cotton (Gossypium hirsutum L.), cowpea (Vignaunguiculata), cucumber (Cucumis sativus), currants and gooseberries(Ribes spp.), eggplant or aubergine (Solanum melongena), eucalyptus(Eucalyptus spp.), flax (Linum usitatissumum L.), geraniums (Pelargoniumspp.), grapefruit (Citrus×paradisi), grapes (Vitus spp.) including winegrapes (Vitus vinifera), guava (Psidium guajava), irises (Iris spp.),lemon (Citrus limon), lettuce (Lactuca sativa), limes (Citrus spp.),maize (Zea mays L.), mango (Mangifera indica), mangosteen (Garciniamangostana), melon (Cucumis melo), millets (Setaria spp, Echinochloaspp, Eleusine spp, Panicum spp., Pennisetum spp.), oats (Avena sativa),oil palm (Ellis quineensis), olive (Olea europaea), onion (Allium cepa),orange (Citrus sinensis), papaya (Carica papaya), peaches and nectarines(Prunus persica), pear (Pyrus spp.), pea (Pisa sativum), peanut (Arachishypogaea), peonies (Paeonia spp.), petunias (Petunia spp.), pineapple(Ananas comosus), plantains (Musa spp.), plum (Prunus domestica),poinsettia (Euphorbia pulcherrima), Polish canola (Brassica rapa),poplar (Populus spp.), potato (Solanum tuberosum), pumpkin (Cucurbitapepo), rice (Oryza sativa L.), roses (Rosa spp.), rubber (Heveabrasiliensis), rye (Secale cereale), safflower (Carthamus tinctorius L),sesame seed (Sesame indium), sorghum (Sorghum bicolor), soybean (Glycinemax L.), squash (Cucurbita pepo), strawberries (Fragaria spp.,Fragaria×ananassa), sugar beet (Beta vulgaris), sugarcanes (Saccharumspp.), sunflower (Helianthus annus), sweet potato (Ipomoea batatas),tangerine (Citrus tangerina), tea (Camellia sinensis), tobacco(Nicotiana tabacum L.), tomato (Lycopersicon esculentum), tulips (Tulipaspp.), turnip (Brassica rapa rapa), walnuts (Juglans spp. L.),watermelon (Citrulus lanatus), wheat (Tritium aestivum), or yams(Discorea spp.).

Embodiment 6. The plant protoplast culture of any one of embodiments 1to 5, wherein the plant protoplast is: (a) encapsulated in a polymer, or(b) encapsulated in a vesicle or liposome, or (c) not encapsulated.

Embodiment 7. The plant protoplast culture of any one of embodiments 1to 6, wherein the culture medium is liquid.

Embodiment 8. The plant protoplast culture of any one of embodiments 1to 7, wherein the culture medium comprises:

-   -   (a) at least 40 millimolar Ca²⁺;    -   (b) at least 50 millimolar Ca²⁺;    -   (c) at least 100 millimolar Ca²⁺;    -   (d) at least 40 millimolar Mg²⁺;    -   (e) at least 50 millimolar Mg²⁺; or    -   (f) at least 100 millimolar Mg²⁺.

Embodiment 9. The plant protoplast culture of any one of embodiments 1to 8, wherein the antioxidant is a low-molecular-weight thiol.

Embodiment 10. The plant protoplast culture of any one of embodiments 1to 8, wherein the antioxidant is glutathione, dithiothreitol,N-acetylcysteine, lipoic acid, ascorbic acid, tocopherols, butylatedhydroxytoluene, butylated hydroxyanisole, or a combination of these.

Embodiment 11. The plant protoplast culture of embodiment 9 or 10,wherein the culture medium comprises at least 1 millimolar glutathione.

Embodiment 12. The plant protoplast culture of any one of embodiments 1to 11, wherein the culture is subjected to hypoxic conditions.

Embodiment 13. The plant protoplast culture of embodiment 12, whereinthe hypoxic conditions comprise:

-   -   (a) about one-half normal atmospheric oxygen concentrations;    -   (b) about 10% oxygen by volume; or    -   (c) about 5% oxygen by volume.

Embodiment 14. The plant protoplast culture of any one of embodiments 1to 13, wherein protoplast viability, when compared to a control plantprotoplast culture without (i) at least 40 millimolar Ca²⁺ or Mg²⁺; (ii)an antioxidant; or (iii) a combination of (i) and (ii), is:

-   -   (a) at least 10% higher after 30 hours' culture;    -   (b) at least 10% higher after 48 hours' culture;    -   (c) at least 10% higher after 72 hours' culture; or    -   (d) at least 10% higher after 96 hours' culture.

Embodiment 15. The plant protoplast culture of any one of embodiments 1to 14, wherein the culture medium comprises at least 100 millimolarCa²⁺, wherein the plant protoplast is obtained from maize, and whereinprotoplast viability, when compared to a control plant protoplastculture without at least 100 millimolar Ca²⁺, is at least 20% higherafter 64 hours' culture.

Embodiment 16. The plant protoplast culture of any one of embodiments 1to 14, wherein the culture medium comprises at least 1 millimolarlow-molecular-weight thiol, wherein the plant protoplast is obtainedfrom maize, and wherein protoplast viability, when compared to a controlplant protoplast culture without at least 1 millimolarlow-molecular-weight thiol, is at least 10% higher after 64 hours'culture.

Embodiment 17. A method of improving viability of a plant protoplast,comprising including in the culture conditions of the protoplast atleast one of:

-   -   (a) at least 40 millimolar Ca²⁺ or Mg²⁺; and    -   (b) at least 1 millimolar low-molecular-weight thiol.

Embodiment 18. The method of embodiment 17, wherein the cultureconditions of the protoplast comprise hypoxic conditions.

Embodiment 19. The method of embodiment 17 or 18, wherein the hypoxicconditions comprise:

-   -   (a) about one-half normal atmospheric oxygen concentrations;    -   (b) about 10% oxygen by volume; or    -   (c) about 5% oxygen by volume.

Embodiment 20. The method of embodiment 17 or 18, wherein the viabilityof a plant protoplast, when compared to that of a control protoplastcultured without at least one of (i) at least 40 millimolar Ca²⁺ orMg²⁺; and (ii) at least 1 millimolar low-molecular-weight thiol, isimproved by:

-   -   (a) at least 10% over at least 24 hours' culture;    -   (b) at least 10% over at least 48 hours' culture;    -   (c) at least 10% over at least 72 hours' culture;    -   (d) at least 10% over at least 96 hours' culture.

Embodiment 21. The method of embodiment 18, wherein the plant protoplastexhibits an improved cell division rate.

Embodiment 22. A protoplast having improved viability, provided by themethod of any one of embodiments 17 to 21.

Embodiment 23. Multiple protoplasts or cells, callus, a somatic embryo,or a regenerated plant, grown from the protoplast of embodiment 22.

Embodiment 24. A composition comprising:

-   -   (a) at least one protoplast having improved viability, provided        by the method of any one of embodiments 17 to 21;    -   (b) at least one effector molecule for inducing a genetic        alteration in the plant cell or plant protoplast, wherein the at        least one effector molecule is selected from the group        consisting of: (i) a polynucleotide selected from the group        consisting of an RNA guide for an RNA-guided nuclease, a DNA        encoding an RNA guide for an RNA-guided nuclease; (ii) a        nuclease selected from the group consisting of an RNA-guided        nuclease, an RNA-guided DNA endonuclease, a type II Cas        nuclease, a Cas9, a type V Cas nuclease, a Cpfl, a CasY, a CasX,        a C2c1, a C2c3, an engineered nuclease, a codon-optimized        nuclease, a zinc-finger nuclease (ZFN), a transcription        activator-like effector nuclease (TAL-effector nuclease),        Argonaute, a meganuclease or engineered meganuclease; or (iii) a        polynucleotide encoding one or more nucleases capable of        effecting site-specific alteration of a target nucleotide        sequence; and    -   (c) optionally, at least one delivery agent selected from the        group consisting of solvents, fluorocarbons, glycols or polyols,        surfactants; primary, secondary, or tertiary amines and        quaternary ammonium salts; organosilicone surfactants; lipids,        lipoproteins, lipopolysaccharides; acids, bases, caustic agents;        peptides, proteins, or enzymes; cell-penetrating peptides; RNase        inhibitors; cationic branched or linear polymers; dendrimers;        counter-ions, amines or polyamines, osmolytes, buffers, and        salts; polynucleotides; transfection agents; antibiotics;        non-specific DNA double-strand-break-inducing agents; and        antioxidants; particles or nanoparticles, magnetic particles or        nanoparticles, abrasive or scarifying agents, needles or        microneedles, matrices, and grids.

Embodiment 25. An array comprising a plurality of containers, eachcomprising at least one protoplast having improved viability, providedby the method of any one of embodiments 17 to 21.

Embodiment 26. A method of improving the cell division rate of a plantprotoplast culture, wherein the culture conditions comprise hypoxicconditions.

Embodiment 27. The method of embodiment 26, wherein the hypoxicconditions comprise:

-   -   (a) about one-half normal atmospheric oxygen concentrations;    -   (b) about 10% oxygen by volume; or    -   (c) about 5% oxygen by volume.

Embodiment 28. The method of embodiment 26 or 27, wherein the cultureconditions further comprise at least 40 millimolar Ca²⁺ or Mg²⁺.

Embodiment 29. A method for making a plant cell having a genomicmodification comprising:

-   -   (a) providing genome editing molecules to a plant cell        previously, concurrently, or subsequently exposed to a hypoxic        condition, a reactive oxygen species (ROS) concentration        lowering agent, or combination thereof; wherein the molecules        comprise: (i) an RNA-guided nuclease and a guide RNA and        optionally a donor template polynucleotide; (ii) a        sequence-specific endonuclease and a donor template        polynucleotide; (iii) one or more polynucleotides encoding an        RNA-guided nuclease and a guide RNA and optionally a donor        template polynucleotide; (iv) a polynucleotide encoding a        sequence-specific endonuclease and a donor template        polynucleotide; or (v) any combination thereof, to modify the        plant cell's genome; and,    -   (b) isolating or propagating a plant cell comprising the genome        modification, thereby making the plant cell having a genomic        modification.

Embodiment 30. The method of embodiment 29, further comprising obtainingcallus, a propagule, or a plant from the isolated or propagated plantcell of step (b) comprising the genome modification, wherein the callus,propagule, or plant comprises a genome modified by the molecule(s) andwherein the propagule is optionally a seed.

Embodiment 31. A method for producing a plant having a genomicmodification comprising:

-   -   (a) providing genome editing molecules to a plant cell        previously, concurrently, or subsequently exposed to a hypoxic        condition, a reactive oxygen species (ROS) concentration        lowering agent, or combination thereof, wherein the molecules        comprise: (i) an RNA-guided nuclease and a guide RNA and        optionally a donor template polynucleotide; (ii) a        sequence-specific endonuclease and a donor template        polynucleotide; (iii) one or more polynucleotides encoding an        RNA-guided nuclease and a guide RNA and optionally a donor        template polynucleotide; (iv) a polynucleotide encoding a        sequence-specific endonuclease and a donor template        polynucleotide; or (v) any combination thereof, to modify the        plant cell's genome;    -   (b) isolating or propagating a plant cell comprising the genome        modification; and,    -   (c) regenerating or obtaining a plant comprising the genome        modification from the plant cell, thereby producing a plant        having the having a genomic modification.

Embodiment 32. The method of embodiment 31, further comprisingharvesting seed from the plant, propagating the plant, or multiplyingthe plant.

Embodiment 33. The method of any one of embodiments 29 to 32, whereinthe ROS concentration lowering agent is an ROS scavenging agent.

Embodiment 34. The method of any one of embodiments 29 to 32, whereinthe plant cell is exposed by: (i) contacting the plant cell with the ROSconcentration lowering agent; or (ii) introducing at least one of theROS concentration lowering agents into the plant cell.

Embodiment 35. The method of any one of embodiments 29 to 32, whereinROS concentration lowering agent is introduced into the plant cell bydirect application, transfection, electroporation, transformation,Agrobacterium-mediated delivery, viral vector mediated delivery, bycontacting or fusing the plant cell with a donor plant cell thatcomprises the agent, by crossing the plant comprising the plant cell toa donor plant that comprises the agent, or any combination thereof.

Embodiment 36. The method of embodiment 35, wherein the donor plant cellor donor plant that comprises the ROS concentration lowering agent isstably or transiently transformed with a polynucleotide encoding theagent.

Embodiment 37. The method of any one of embodiments 29 to 32, whereinthe genome editing molecule(s) are provided to the plant cell byintroducing the molecule(s) into the plant cell by direct application,transfection, electroporation, transformation, Agrobacterium-mediateddelivery, viral vector mediated delivery, by fusing or contacting theplant cell with another plant cell that comprises the agent, by crossingthe plant comprising the plant cell to a plant that comprises themolecules, or any combination thereof.

Embodiment 38. The method of any one of embodiments 29 to 32, whereinthe plant cell exposed to the hypoxiccondition or ROS concentrationlowering agent: (i) contains the RNA-guided nuclease or contains one ormore polynucleotides encoding an RNA-guided nuclease and is associatedwith and/or contacts the guide RNA; or (ii) contains thesequence-specific endonuclease or contains one or more polynucleotidesencoding a sequence-specific endonuclease and is associated with and/orcontacts the donor template polynucleotide.

Embodiment 39. A system for producing a plant cell having a genomicmodification comprising: (a) a plant cell subjected to a hypoxiccondition, or treated with a reactive oxygen species (ROS) scavengingagent, or both subjected to the hypoxic condition and treated with theROS scavenging agent; and (b) genome editing molecule(s) comprising: (i)an RNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (ii) a sequence-specific endonuclease and a donortemplate polynucleotide; (iii) one or more polynucleotides encoding anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (iv) one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof; wherein the plant cell is associated with,contacts, and/or contains the molecule(s).

Embodiment 40. A system for producing a plant cell having a genomicmodification comprising: (a) a plant cell wherein a reactive oxygenspecies (ROS) concentration is lowered in comparison to a control plantcell; and (b) genome editing molecule(s) comprising: (i) an RNA-guidednuclease and a guide RNA and optionally a donor template polynucleotide;(ii) a sequence-specific endonuclease and a donor templatepolynucleotide; (iii) one or more polynucleotides encoding an RNA-guidednuclease and a guide RNA and optionally a donor template polynucleotide;(iv) one or more polynucleotide(s) encoding a sequence-specificendonuclease and a donor template polynucleotide; or (v) any combinationthereof; wherein the plant cell is associated with, contacts, and/orcontains the molecule(s).

Embodiment 41. The system of embodiment 40, wherein the ROSconcentration is lowered by treating the cell with an exogenouslyprovided ROS scavenging agent and/or subjecting the cell to a hypoxiccondition.

Embodiment 42. A composition comprising: (a) (i) a plant cell subjectedto a hypoxic condition, or treated with an exogenous reactive oxygenspecies (ROS) scavenging agent, or both subjected to the hypoxiccondition and treated with the ROS scavenging agent; or (ii) a plantcell or plant cell subjected to a hypoxic condition and an exogenous ROSscavenging agent; and (b) genome editing molecule(s) comprising: (i) anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (ii) a sequence-specific endonuclease and a donortemplate polynucleotide; (iii) one or more polynucleotides encoding anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (iv) one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof; wherein the plant cell is associated with,contacts, and/or contains the molecule(s).

Embodiment 43. A composition comprising: (a) a plant cell wherein areactive oxygen species (ROS) concentration is lowered in comparison toa control plant cell; and (b) genome editing molecule(s) comprising: (i)an RNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (ii) a sequence-specific endonuclease and a donortemplate polynucleotide; (iii) one or more polynucleotides encoding anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (iv) one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof; wherein the plant cell is associated with,contacts, and/or contains the agent and the molecule(s).

Embodiment 44. The composition of embodiment 43, wherein the ROSconcentration is lowered by treating the cell with an exogenous ROSscavenging agent, by subjecting the cell to a hypoxic condition, orwherein the composition or plant cell comprises an exogenous ROSscavenging agent.

Embodiment 45. The method of any one of embodiments 29 to 32, system ofembodiment 39 or 41, or composition of embodiment 42 or 44, wherein theROS concentration lowering agent or ROS scavenging agent comprises anon-enzymatic ROS scavenging agent.

Embodiment 46. The method, system or composition of embodiment 45,wherein the non-enzymatic ROS scavenging agent is ascorbic acid, alow-molecular-weight thiol, a pro-thiol, a tocopherol, a carotenoid, aflavonoid, or combination thereof.

Embodiment 47. The method of any one of embodiments 29 to 32, system ofembodiment 39 or 41, or composition of embodiment 42 or 44, wherein theROS concentration lowering agent or the ROS scavenging agent comprisesan enzymatic ROS scavenging agent.

Embodiment 48. The method of embodiment 47, wherein the enzymatic ROSscavenging agent comprises a catalase, an ascorbate peroxidase, adehydroascorbate reductase, guaiacol peroxidase, monodehydroascorbatereductase, a peroxidase, a superoxide dismutase, or a combinationthereof.

Embodiment 49. The method of any one of embodiments 29 to 32, system ofembodiment 39 or 41, or composition of embodiment 42 or 44, wherein theROS concentration lowering agent or the ROS scavenging agent comprises acombination of at least one enzymatic and at least one non-enzymatic ROSscavenging agent.

Embodiment 50. The method of any one of embodiments 29 to 32, system ofembodiment 39 or 41, or composition of embodiment 42 or 44, wherein thecell comprises an exogenous polynucleotide that produces the ROSconcentration lowering agent or ROS scavenging agent in the cell.

Embodiment 51. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein: (i) the hypoxic condition comprisesmaintaining the cell in air comprising an oxygen concentration of about12% to about 5% oxygen by volume; or (ii) the ROS concentration islowered by maintaining the cell in air comprising an oxygenconcentration of about 12% to about 5% oxygen by volume.

Embodiment 52. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the cell is in a liquid culture media andwherein the hypoxic growth condition or hypoxic condition comprisesmaintaining the cell and the media in air comprising an oxygenconcentration of about 12% to about 5% oxygen by volume or wherein theROS level is lowered by maintaining the cell and the media in aircomprising an oxygen concentration of about 12% to about 5% oxygen byvolume.

Embodiment 53. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the hypoxic condition comprisesmaintaining the cell in a liquid culture media having a dissolved oxygenconcentration that is lowered in comparison to a dissolved oxygenconcentration of liquid culture media kept under an oxygen concentrationof 20% by volume or wherein the ROS concentration is lowered bymaintaining the cell in a liquid culture media having a dissolved oxygenconcentration that is lowered in comparison to a dissolved oxygenconcentration of liquid culture media kept under an oxygen concentrationof 20% by volume.

Embodiment 54. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the hypoxic condition or hypoxic conditionis induced by treating the cell with a hypoxia mimetic, wherein the ROSconcentration is lowered by treating the cell with a hypoxia mimetic, orwherein the composition comprises an exogenous hypoxia mimetic.

Embodiment 55. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the concentration of more than one ROS islowered.

Embodiment 56. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the ROS is hydrogen peroxide, a superoxideradical, a peroxide ion, a hydroperoxyl radical, or a hydroxyl radical.

Embodiment 57. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the plant cell is in culture media, in aplant, or in a plant tissue.

Embodiment 58. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the plant cell is part of a callusculture, an embryogenic callus culture, or an embryo.

Embodiment 59. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the plant cell is a plant protoplast, amature pollen cell, a microspore, or a megaspore.

Embodiment 60. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the genome editing molecule(s) can providefor a substitution or deletion of a single nucleotide residue in anendogenous gene of the plant cell.

Embodiment 61. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein frequency of the genome modification isincreased in comparison to a control method wherein a control plant cellis not exposed to a ROS concentration lowering agent, a ROS scavengingagent, or a hypoxic condition.

Embodiment 62. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein a frequency of homology directed repair(HDR) of a target gene in the plant cell is increased by at least1.1-fold in comparison to a control composition comprising the genomeediting molecules, wherein: (i) a control plant cell of the controlcomposition is not subjected to the hypoxic condition, is not treatedwith the ROS scavenging agent, or wherein the ROS scavenging agent isabsent; or (ii) ROS concentrations are not lowered in a control plantcell of the control composition.

Embodiment 63. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the plant cell is a monocot plant cell.

Embodiment 64. The method, system, or composition of embodiment 63,wherein the monocot plant cell is a barley, maize, millet, oat, rice,rye, sorghum, or wheat plant cell.

Embodiment 65. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the plant cell is a dicot plant cell.

Embodiment 66. The method, system, or composition of embodiment 65,wherein the dicot plant cell is an alfalfa, canola, oilseed rape,cotton, flax, potato, soybean, or tomato plant cell.

Embodiment 67. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the RNA-guided nuclease comprises anRNA-guided DNA endonuclease, a type II Cas nuclease, a Cas9 nuclease, atype V Cas nuclease, a Cpfl nuclease, a CasY nuclease, a CasX nuclease,a C2c1 nuclease, a C2c3 nuclease, or an engineered nuclease.

Embodiment 68. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the sequence-specific endonucleasecomprises a zinc-finger nuclease (ZFN), a transcription activator-likeeffector nuclease (TAL-effector nuclease), Argonaute, a meganuclease, orengineered meganuclease.

Embodiment 69. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the ROS concentration lowering agent orthe ROS scavenging agent is heterologous to the plant cell and/orwherein the genome editing molecule(s) are heterologous to the plantcell.

Embodiment 70. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one ofembodiments 42 to 44, wherein the ROS concentration lowering agent is anexogenous ROS concentration lowering agent or the ROS scavenging agentis an exogenous ROS scavenging agent and/or wherein the genome editingmolecule(s) are exogenous genome editing molecule(s).

Embodiment 71. The method of any one of embodiments 29 to 32, system ofany one of embodiments 39 to 41, or composition of any one of claims 42to 44, wherein Ca²⁺ and/or Mg²⁺ are provided at a concentration of about40 mM to 150 mM.

Embodiment 72. A plant cell culture comprising:

-   -   (a) a plant cell culture medium;    -   (b) a plant cell exposed to a hypoxic condition, or to a        reactive oxygen species (ROS) concentration lowering agent, or        to a combination thereof, wherein the plant cell is contained or        supported by the plant cell culture medium; and,    -   (c) genome editing molecule(s) comprising: (i) an RNA-guided        nuclease and a guide RNA and optionally a donor template        polynucleotide; (ii) a sequence-specific endonuclease and a        donor template polynucleotide; (iii) one or more polynucleotides        encoding an RNA-guided nuclease and a guide RNA and optionally a        donor template polynucleotide; (iv) one or more        polynucleotide(s) encoding a sequence-specific endonuclease and        a donor template polynucleotide; or (v) any combination thereof;        wherein the plant cell is associated with, contacts, and/or        contains the molecule(s).

Embodiment 73. The plant cell culture of embodiment 72, wherein thehypoxic condition comprises exposing the plant cell to an atmospherecomprising an oxygen concentration of about 12% to about 5% oxygen byvolume.

Embodiment 74. The plant cell culture of embodiment 72 or 73, whereinthe ROS concentration lowering agent is a ROS scavenging agent.

Embodiment 75. The plant cell culture of embodiment 74, wherein the ROSscavenging agent is ascorbic acid, a low-molecular-weight thiol, apro-thiol, a tocopherol, a carotenoid, a flavonoid, or combinationthereof.

Embodiment 76. The plant cell culture of any one of embodiments 72, 73,74, or 75, wherein a frequency of homology directed repair (HDR) of atarget gene in the plant cell is increased by at least 2-fold incomparison to a control plant cell culture provided with the genomeediting molecules and plant cells not exposed to a hypoxic condition, orto a reactive oxygen species (ROS) concentration lowering agent, or to acombination thereof.

Embodiment 77. The plant cell culture of any one of embodiments 72, 73,74, 75, or 76, wherein Ca²⁺ and/or Mg²⁺ is provided at a concentrationof about 40 mM to 150 mM in the plant cell culture medium.

Embodiment 78. A method for making a plant cell having a genomicmodification comprising:

-   -   (a) providing genome editing molecules to a plant cell        previously, concurrently, or subsequently subjected to a hypoxic        condition, or to a reactive oxygen species (ROS) concentration        lowering agent, or to a combination thereof; wherein the        molecules comprise: (i) an RNA-guided nuclease and a guide RNA        and optionally a donor template polynucleotide; (ii) a        sequence-specific endonuclease and a donor template        polynucleotide; (iii) one or more polynucleotides encoding an        RNA-guided nuclease and a guide RNA and optionally a donor        template polynucleotide; (iv) a polynucleotide encoding a        sequence-specific endonuclease and a donor template        polynucleotide; or (v) any combination thereof, to modify the        plant cell's genome; and,    -   (b) isolating, selecting, identifying, and/or propagating a        plant cell comprising the genome modification, thereby making        the plant cell having a genomic modification.

Embodiment 79. The method of embodiment 78, further comprising obtainingcallus, a propagule, or a plant from the isolated, selected, identified,and/or propagated plant cell of step (b) comprising the genomemodification, wherein the callus, propagule, or plant comprises a genomemodified by the molecule(s) and wherein the propagule is optionally aseed.

Embodiment 80. The method of embodiment 78 or 79, wherein the hypoxiccondition comprises maintaining a plant, plant tissue, plant part, orplant cell culture containing the plant cell under an atmospherecomprising an oxygen concentration of about 12% to about 5% oxygen byvolume.

Embodiment 81. The method of any one of embodiments 78, 79, or 80,wherein the ROS concentration lowering agent is a ROS scavenging agent.

Embodiment 82. The method of embodiment 81, wherein the ROS scavengingagent is ascorbic acid, a low-molecular-weight thiol, a pro-thiol, atocopherol, a carotenoid, a flavonoid, or combination thereof.

Embodiment 83. The method of any one of embodiments 78, 79, 80, 81, or82, wherein a frequency of homology directed repair (HDR) of a targetgene in the plant cell of step (a) is increased by at least 2-fold incomparison to a control plant cell provided with the genome editingmolecules and is not exposed to a hypoxic condition, or to a reactiveoxygen species (ROS) concentration lowering agent, or to a combinationthereof.

Embodiment 84. The method of any one of embodiments 78, 79, 80, 81, 82,or 83, wherein the plant cell in at least step (a) is contained orsupported by a plant cell culture medium and Ca²⁺ and/or Mg²⁺ isprovided in the plant cell culture medium at a concentration of about 40mM to 150 mM.

Embodiment 85. The method of any one of embodiments 78, 79, 80, 81, 82,83, or 84, wherein: (i) the hypoxic condition comprises maintaining aplant, plant tissue, plant part, or plant cell culture containing theplant cell under an atmosphere comprising an oxygen concentration ofabout 12% to about 5% oxygen by volume; (ii) the ROS lowering agent isascorbic acid, a low-molecular-weight thiol, a pro-thiol, a tocopherol,a carotenoid, a flavonoid, or combination thereof; and/or (iii) Ca²⁺and/or Mg²⁺ is provided at a concentration of about 40 mM to 150 mM tothe plant cell in at least step (a).

Embodiment 86. A system for producing a plant cell having a genomicmodification comprising: (a) a plant cell subjected to a hypoxiccondition, or treated with a reactive oxygen species (ROS) scavengingagent, or both subjected to the hypoxic condition and treated with theROS scavenging agent; and (b) genome editing molecule(s) comprising: (i)an RNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (ii) a sequence-specific endonuclease and a donortemplate polynucleotide; (iii) one or more polynucleotides encoding anRNA-guided nuclease and a guide RNA and optionally a donor templatepolynucleotide; (iv) one or more polynucleotide(s) encoding asequence-specific endonuclease and a donor template polynucleotide; or(v) any combination thereof; wherein the plant cell is associated with,contacts, and/or contains the molecule(s).

Embodiment 87. The system of embodiment 86, wherein the hypoxiccondition comprises exposing a plant, plant tissue, plant part, or plantcell culture containing the plant cell to an oxygen concentration ofabout 12% to about 5% oxygen by volume.

Embodiment 88. The system of embodiment 86 or 87, wherein the ROSconcentration lowering agent is an ROS scavenging agent.

Embodiment 89. The system of any one of embodiments 86, 87, or 88,wherein the ROS scavenging agent is ascorbic acid, alow-molecular-weight thiol, a pro-thiol, a tocopherol, a carotenoid, aflavonoid, or combination thereof.

Embodiment 90. The system of any one of embodiments 86, 87, 88, or 89,wherein a frequency of homology directed repair (HDR) of a target genein the plant cell is increased by at least 1.1-fold in comparison to acontrol plant cell culture comprising the genome editing moleculeswherein a control plant cell is not subjected to a hypoxic growthcondition and/or a ROS scavenging agent is absent.

Embodiment 91. The system of any one of embodiments 86, 87, 88, 89, or90, wherein the plant cell is contained or supported by a plant cellculture medium and Ca²⁺ and/or Mg²⁺ is provided at a concentration ofabout 40 mM to 150 mM in the plant cell culture medium.

EXAMPLES Example 1

This example illustrates techniques for preparing a plant cell or plantprotoplast useful in compositions and methods of the disclosure. Morespecifically this non-limiting example describes techniques forpreparing isolated, viable plant protoplasts from monocot and dicotplants.

The following mesophyll protoplast preparation protocol (modified fromone publicly available atmolbio[dot]mgh[dot]harvard.edu/sheenweb/protocols_reg[dot]html) isgenerally suitable for use with monocot plants such as maize (Zea mays)and rice (Oryza sativa):

Prepare an enzyme solution containing 0.6 molar mannitol, 10 millimolarMES pH 5.7, 1.5% cellulase R10, and 0.3% macerozyme R10. Heat the enzymesolution at 50-55 degrees Celsius for 10 minutes to inactivate proteasesand accelerate enzyme solution and cool it to room temperature beforeadding 1 millimolar CaCl₂, 5 millimolar β-mercaptoethanol, and 0.1%bovine serum albumin. Pass the enzyme solution through a 0.45 micrometerfilter. Prepare a washing solution containing 0.6 molar mannitol, 4millimolar MES pH 5.7, and 20 millimolar KCl.

Obtain second leaves of the monocot plant (e. g., maize or rice) and cutout the middle 6-8 centimeters. Stack ten leaf sections and cut into 0.5millimeter-wide strips without bruising the leaves. Submerge the leafstrips completely in the enzyme solution in a petri dish, cover withaluminum foil, and apply vacuum for 30 minutes to infiltrate the leaftissue. Transfer the dish to a platform shaker and incubate for anadditional 2.5 hours' digestion with gentle shaking (40 rpm). Afterdigestion, carefully transfer the enzyme solution (now containingprotoplasts) using a serological pipette through a 35 micrometer nylonmesh into a round-bottom tube; rinse the petri with 5 milliliters ofwashing solution and filter this through the mesh as well. Centrifugethe protoplast suspension at 1200 rpm, 2 minutes in a swing-bucketcentrifuge. Aspirate off as much of the supernatant as possible withouttouching the pellet; gently wash the pellet once with 20 milliliterswashing buffer and remove the supernatant carefully. Gently resuspendthe pellet by swirling in a small volume of washing solution, thenresuspend in 10-20 milliliters of washing buffer. Place the tube uprighton ice for 30 minutes-4 hours (no longer). After resting on ice, removethe supernatant by aspiration and resuspend the pellet with 2-5milliliters of washing buffer. Measure the concentration of protoplastsusing a hemocytometer and adjust the concentration to 2×10{circumflexover ( )}5 protoplasts/milliliter with washing buffer.

The following mesophyll protoplast preparation protocol (modified fromone described by Niu and Sheen (2012) Methods Mol. Biol., 876:195-206,doi: 10.1007/978-1-61779-809-2_16) is generally suitable for use withdicot plants such as Arabidopsis thaliana and brassicas such as kale(Brassica oleracea).

Prepare an enzyme solution containing 0.4 M mannitol, 20 millimolar KCl,20 millimolar MES pH 5.7, 1.5% cellulase R10, and 0.4% macerozyme R10.Heat the enzyme solution at 50-55 degrees Celsius for 10 minutes toinactivate proteases and accelerate enzyme solution, and then cool it toroom temperature before adding 10 millimolar CaCl₂), 5 millimolarβ-mercaptoethanol, and 0.1% bovine serum albumin. Pass the enzymesolution through a 0.45 micrometer filter. Prepare a “W5” solutioncontaining 154 millimolar NaCl, 125 millimolar CaCl₂, 5 millimolar KCl,and 2 millimolar MES pH 5.7. Prepare a “MMg solution” solutioncontaining 0.4 molar mannitol, 15 millimolar MgCl₂, and 4 millimolar MESpH 5.7.

Obtain second or third pair true leaves of the dicot plant (e. g., abrassica such as kale) and cut out the middle section. Stack 4-8 leafsections and cut into 0.5 millimeter-wide strips without bruising theleaves. Submerge the leaf strips completely in the enzyme solution in apetri dish, cover with aluminum foil, and apply vacuum for 30 minutes toinfiltrate the leaf tissue. Transfer the dish to a platform shaker andincubate for an additional 2.5 hours' digestion with gentle shaking (40rpm). After digestion, carefully transfer the enzyme solution (nowcontaining protoplasts) using a serological pipette through a 35micrometer nylon mesh into a round-bottom tube; rinse the petri dishwith 5 milliliters of washing solution and filter this through the meshas well. Centrifuge the protoplast suspension at 1200 rpm, 2 minutes ina swing-bucket centrifuge. Aspirate off as much of the supernatant aspossible without touching the pellet; gently wash the pellet once with20 milliliters washing buffer and remove the supernatant carefully.Gently resuspend the pellet by swirling in a small volume of washingsolution, then resuspend in 10-20 milliliters of washing buffer. Placethe tube upright on ice for 30 minutes-4 hours (no longer). Afterresting on ice, remove the supernatant by aspiration and resuspend thepellet with 2-5 milliliters of MMg solution. Measure the concentrationof protoplasts using a hemocytometer and adjust the concentration to2×10{circumflex over ( )}5 protoplasts/milliliter with MMg solution.

Example 2

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes media and culture conditions forimproving viability of isolated plant protoplasts.

Table 1 provides the compositions of different liquid basal mediasuitable for culturing plant cells or plant protoplasts; final pH of allmedia was adjusted to 5.8 if necessary.

TABLE 1* Concentration (mg/L unless otherwise noted) YPIM Component SH8p PIM P2 B- Casamino acids 250 Coconut water 20000 Ascorbic acid 2biotin 0.01 0.01 Cholicalciferol (Vitamin D-3) 0.01 choline chloride 1Citric acid 40 Cyanocobalamin (Vitamin B-12) 0.02 D-calcium pantothenate1 1 D-Cellobiose 250 D-Fructose 250 D-Mannose 250 D-Ribose 250D-Sorbitol 250 D-Xylose 250 folic acid 0.4 0.2 Fumaric acid 40 L-Malicacid 40 L-Rhamnose 250 p-Aminobenzoic acid 0.02 Retinol (Vitamin A) 0.01Riboflavin 0.2 Sodium pyruvate 20 2,4-D 0.5 0.2 1 5 16-benzylaminopurine (BAP) 1 Indole-3-butyric acid (IBA) 2.5 Kinetin 0.1Naphthaleneacetic acid (NAA) 1 parachlorophenoxyacetate (pCPA) 2Thidiazuron 0.022 Zeatin 0.5 AlCl3 0.03 Bromocresol purple 8 CaCl₂•2H₂O200 600 440 200 440 CoCl₂•6H₂O 0.1 0.025 0.1 CuSO₄•5H₂O 0.2 0.025 0.030.2 0.03 D-Glucose 68400 40000 40000 D-Mannitol 52000 250 60000 5200060000 FeSO₄•7H₂O 15 27.8 15 15 15 H₃BO₃ 5 3 1 5 1 KCl 300 KH₂PO₄ 170 170170 KI 1 0.75 0.01 1 0.01 KNO₃ 2500 1900 505 2500 505 MES pH 5.8 (mM)3.586 25 25 MgSO₄•7H₂O 400 300 370 400 370 MnSO₄•H₂O 10 10 0.1 10 0.1Na₂EDTA 20 37.3 20 20 20 Na₂MoO₄•2H₂O 0.1 0.25 0.1 NH₄H₂PO₄ 300 300NH₄NO₃ 600 160 160 NiCl₂•6H₂O 0.03 Sucrose 30000 2500 30000 ZnSO₄•7H₂0 12 1 1 1 Tween-80 (microliter/L) 10 10 Inositol 1000 100 100 1000 100Nicotinamide 1 Nicotinic acid 5 1 5 1 Pyridoxine•HCl 0.5 1 1 0.5 1Thiamine•HCl 5 1 1 5 1 *Sources for basal media: SH—Schenk andHildebrandt, Can. J. Bot. 50: 199 (1971). 8p—Kao and Michayluk, Planta126: 105 (1975). P2—SH but with hormones from Potrykus et al., Mol. Gen.Genet. 156: 347 (1977). PIM—Chupeau et al., The Plant Cell 25: 2444(2013).

Example 3

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes methods for encapsulating isolated plantprotoplasts.

When protoplasts are encapsulated in alginate or pectin, they remainintact far longer than they would in an equivalent liquid medium. Inorder to encapsulate protoplasts, a liquid medium (“calcium base”) isprepared that is in all other respects identical to the final desiredrecipe with the exception that the calcium (usually CaCl₂.2H₂O) isincreased to 80 millimolar. A second medium (“encapsulation base”) isprepared that has no added calcium but contains 10 g/L of theencapsulation agent, e. g., by making a 20 g/L solution of theencapsulation agent and adjusting its pH with KOH or NaOH until it isabout 5.8, making a 2× solution of the final medium (with no calcium),then combining these two solutions in a 1:1 ratio. Encapsulation agentsinclude alginate (e. g., alginic acid from brown algae, catalogue numberA0682, Sigma-Aldrich, St. Louis, Mo.) and pectin (e. g., pectin fromcitrus peel, catalogue number P9136, Sigma-Aldrich, St. Louis, Mo.;various pectins including non-amidated low-methoxyl pectin, cataloguenumber 1120-50 from Modernist Pantry, Portsmouth, N.H.). The solutions,including the encapsulation base solution, is filter-sterilized througha series of filters, with the final filter being a 0.2-micrometerfilter. Protoplasts are pelleted by gentle centrifugation andresuspended in the encapsulation base; the resulting suspension is addeddropwise to the calcium base, upon which the protoplasts are immediatelyencapsulated in solid beads.

Example 4

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes observations of effects on protoplastviability obtained by adding non-conventionally high levels of divalentcations to culture media.

Typical plant cell or plant protoplast media contain between or about 2to about 4 millimolar calcium cations and between or about 1-1.5millimolar magnesium cations. In the course of experiments varying andadding components to media, it was discovered that the addition ofnon-conventionally high levels of divalent cations had a surprisinglybeneficial effect on plant cell or plant protoplast viability.Beneficial effects on plant protoplast viability begin to be seen whenthe culture medium contains about 30 millimolar calcium cations (e. g.,as calcium chloride) or about 30 millimolar magnesium cations (e. g., asmagnesium chloride). Even higher levels of plant protoplast viabilitywere observed with increasing concentrations of calcium or magnesiumcations, i. e., at about 40 millimolar or about 50 millimolar calcium ormagnesium cations. The result of several titration experiments indicatedthat greatest improvement in protoplast viability was seen using mediacontaining between or about 50 to about 100 millimolar calcium cationsor 50 to about 100 millimolar magnesium cations; no negative effects onprotoplast viability or physical appearance was observed at these highcation levels. This was observed in multiple experiments usingprotoplasts obtained from several plant species including maize(multiple germplasms, e. g., B73, A188, B104, HiIIA, HiIIB, BMS), rice,wheat, soy, kale, and strawberry; improved protoplast viability wasobserved in both encapsulated protoplasts and non-encapsulatedprotoplasts. Addition of potassium chloride at the same levels had noeffect on protoplast viability. It is possible that inclusion ofslightly lower (but still non-conventionally high) levels of divalentcations (e. g., about 10 millimolar, about 15 millimolar, about 20millimolar, or about 25 millimolar calcium cations or magnesium cations)in media is beneficial for plant cells or plant protoplasts ofadditional plant species.

Example 5

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes observations of effects on maizeprotoplast viability obtained by adding non-conventionally high levelsof divalent cations to culture media. Separate suspensions of maize B73and A188 protoplasts (2×10{circumflex over ( )}5 cells per milliliter)were prepared in YPIM B-liquid medium containing various combinations ofthe added salts calcium chloride, potassium ascorbate, and magnesiumchloride or magnesium sulfate. One-half milliliter aliquots of thesuspensions were dispensed into a 24-well microtiter plate in thearrangement shown in Table 2, which lists the concentrations of calciumchloride (“Ca”), potassium ascorbate (“A”), and magnesium chloride(“MgCl2”) or magnesium sulfate (“MgSO4”) in millimolar values.

TABLE 2 YPIM B- Ca = 0, A = 0.1 Ca = 0, A = 0.2 Ca = 0, A = 0.5 Ca = 0,A = 1 YPIM B- Ca = 50, A = 0 Ca = 50, A = 0.1 Ca = 50, A = 0.2 Ca = 50,A = 0.5 Ca = 50, A = 1 YPIM B- Ca = 100, A = 0 Ca = 100, A = 0.1 Ca =100, A = 0.2 Ca = 100, A = 0.5 Ca = 100, A = 1 YPIM B- YPIM B- MgCl₂ =50 MgCl₂ = 100 MgSO₄ = 50 MgSO₄ = 100 YPIM B-

Viability was judged by Evans blue staining and visualization under alight microscope. After 96 hours, both maize species were still highlyviable in all wells. After 288 hours, there were clear differences atvarious calcium and magnesium concentrations, but only slight effects atvarious ascorbate concentrations.

The observations at 288 hours were recorded as follows: Maize B73:protoplasts in all Ca=0 wells appeared small and dead; protoplasts inCa=50 wells appeared larger but were now also almost all dead;protoplasts in Ca=100 wells still appeared larger and had a viability ofbetween or 10-20%. Protoplasts in MgCl2=50 wells were similar to thosein Ca=100 wells, and protoplasts in MgCl2=100 wells had much higherviability than any well. Wells with MgSO4=50 or 100 showed only a modestimprovement in protoplast viability. Maize A188: protoplasts in all Ca=0wells appeared small and dead; protoplasts in Ca=50 wells appeared andhad about 20% viability; protoplasts in Ca=100 wells had about 70%viability and were visibly healthier. Addition of ascorbate at 0.2millimolar and above to the wells with added calcium appeared toslightly decrease viability. Wells with MgSO4=50 had about 30-40%viability, and wells with MgCl2=100 had about 70% viability. Wells withMgSO4=50 or 100 showed only a modest improvement in protoplastviability. These results demonstrate that calcium chloride or magnesiumchloride added at non-conventionally high levels improved maizeprotoplast viability over a culture time of ˜12 days.

Example 6

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes observations of effects on maize,soybean, and strawberry protoplast viability obtained by addingnon-conventionally high levels of divalent cations to culture media.Separate suspensions of maize B73, winter wheat, soy, and strawberryprotoplasts (2×10{circumflex over ( )}5 cells per milliliter) wereprepared in YPIM B-liquid medium containing calcium chloride at 0, 50,or 100 millimolar. One-half milliliter aliquots of the suspensions weredispensed into a 24-well microtiter plate.

Viability at day 8 of culture was judged by visualization under a lightmicroscope. At this point, the viability of the maize protoplasts in the0, 50, and 100 millimolar calcium conditions was 10%, 30%, and 80%,respectively. There were no large differences observed at this timepoint for protoplasts of the other species.

Viability at day 13 was judged by Evans blue staining and visualizationunder a light microscope. At this point, the viability of the maizeprotoplasts in the 0, 50, and 100 millimolar calcium conditions was 0%,0%, and 10%, respectively; viability of the soybean protoplasts in the0, 50, and 100 millimolar calcium conditions was 0%, 50%, and 50%,respectively; and viability of the maize protoplasts in the 0 and 50millimolar calcium conditions was 0% and 50%, respectively (viabilitywas not measured for the 100 millimolar condition). These resultsdemonstrate that culture conditions including calcium cations at 50 or100 millimolar improved viability of both monocot and dicot protoplastsover a culture time of ˜13 days.

Example 7

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes observations of effects on maizeprotoplast viability obtained by adding non-conventionally high levelsof divalent cations to culture media. Separate suspensions of maize A188protoplasts (2×10{circumflex over ( )}5 cells per milliliter) wereprepared in YPIM B-liquid medium containing calcium chloride at 0 or 50millimolar. One-half milliliter aliquots of the suspensions weredispensed into a 24-well microtiter plate.

Viability was judged by visualization under a light microscope. At 96hours, protoplasts grown with 50 millimolar calcium cations appearedhealthier than those grown with no added calcium. At 168 hours (7 days),wells with 50 millimolar calcium cations still contained very manylarge, healthy-looking protoplasts, whereas protoplasts in the wellswith no added calcium were nearly all dead. This experiment was carriedon to day 20, at which point the protoplasts in the wells with 50millimolar calcium had generated cell walls and undergone at least somecell division. These results demonstrate that culture conditionsincluding calcium cations at 50 millimolar improved viability, cell wallregeneration, and cell division of maize protoplasts over a culture timeof at least 7 to 20 days.

Example 8

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes observations of effects on maizeprotoplast viability obtained by adding non-conventionally high levelsof divalent cations to culture media.

Separate suspensions of maize B73 protoplasts (2×10{circumflex over( )}5 cells per milliliter) were prepared in PIM B-liquid medium(identical to YPIM B-medium except with the 6-benzylaminopurinesubstituted with 0.022 milligrams/L thidiazuron) containing calciumchloride added at 0, 5, 20, 40, 70, or 100 millimolar. One-halfmilliliter aliquots of the suspensions were dispensed into a 24-wellmicrotiter plate.

Viability was judged by visualization under a light microscope at day 7and at day 14 of culture. In this experiment, by day 7 the maizeprotoplasts were dead in the wells containing less than 40 millimolarcalcium; the maize protoplasts in the wells containing 40, 70, or 100millimolar calcium formed clusters of viable, healthy cells with celldivision occurring, with the strongest enhanced viability and celldivision observed at 100 millimolar calcium. These results demonstratethat culture conditions including calcium cations at 40, 70, or 100improved viability, cell wall regeneration, and cell division of maizeprotoplasts over a culture time of at least 7 to 14 days.

Example 9

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes observations of effects on maizeprotoplast viability obtained by adding non-conventionally high levelsof divalent cations to culture media. Separate suspensions of maize B73and A188 protoplasts (2×10{circumflex over ( )}5 cells per milliliter)were prepared in PIM B-liquid medium (identical to YPIM B-medium exceptwith the 6-benzylaminopurine substituted with 0.022 milligrams/Lthidiazuron) containing calcium chloride added at 0 or 50 millimolar.One-half milliliter aliquots of the suspensions were dispensed into a24-well microtiter plate.

Viability was judged by visualization under a light microscope. In thisexperiment, by day 6 the maize A188 protoplasts were about 40% viable inthe wells containing no added calcium but showed much higher viabilityin the wells containing 50 millimolar calcium, where several wellsshowed 100% viability. The maize B73 protoplasts in the wells containingno added calcium had all died, but wells containing 50 millimolarcalcium still contained viable cells.

Example 10

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes observations of effects on maizeprotoplast viability obtained by adding non-conventionally high levelsof divalent cations or a low-molecular-weight antioxidant to culturemedia. Separate suspensions of maize B73 and A188 protoplasts(2×10{circumflex over ( )}5 cells per milliliter) were prepared in YPIMB-liquid medium containing (a) calcium chloride added at 100 millimolar,or (b) 1 millimolar glutathione, or (c) no added calcium or glutathione.One-milliliter aliquots of the suspensions were dispensed into a 24-wellmicrotiter plate. At 16, 40, 64, and 136 hours of culture, 50-microlitersamples were taken for hemocytometer analysis from each well; for theplates containing maize A188 protoplasts, parallel 50-microliter sampleswere taken from a replicate well at 16, 40, and 64 hours of culture forquantification using a Cellometer cell counter (Nexcelom Bioscience LLC,Lawrence, Mass.).

Viability was determined by Evans blue staining and quantification usinga hemocytometer. Under conditions with high concentrations of calcium,Evans blue can create precipitates that interfere with cell counting; toprevent this, 5 microliters of an EDTA solution was added to the samplesfrom the wells containing 100 millimolar calcium chloride immediatelyprior to staining. Results from the hemocytometer analysis are providedin Table 3 (results from the Cellometer analysis were very similar);“Control”=YPIM B-medium with no added calcium or glutathione. Theseresults demonstrate that inclusion in the medium of eithernon-conventionally high (100 millimolar) calcium cations or thelow-molecular-weight thiol antioxidant glutathione resulted inincreasing protoplast viability of both maize lines by (a) at least 10%higher after 30 hours (in this example, about 10-34% higher at 40 hours)culture; (b) at least 10% higher after 48 hours' culture hours (in thisexample, between 17-53% higher at 64 hours); or (c) at least 10% higherafter 72 hours' culture hours or at least 10% higher after 96 hours'culture hours (in this example, about 12-at least 46% higher at 138hours).

TABLE 3 Cell Viability (%) Type Hours Control 100 mM Ca 1 mM GSH B73  090 90 90  16 65 65 77  40 38 57 72  64 31 58 48 136 12 30 24 A188  0 9090 90  16 60 67 69  40 40 57 50  64  6 59 50 136  0 46 42

Example 11

This example illustrates culture conditions effective in improvingviability of plant cells or plant protoplasts. More specifically, thisnon-limiting example describes the effects on maize protoplast viabilityobtained by adding non-conventionally high levels of divalent cations toculture media. Separate suspensions of maize protoplasts from fivedifferent germplasm lines (A188, B73, B104, HiIIA, HiIIB)(2×10{circumflex over ( )}5 cells per milliliter) were prepared in YPIMB-liquid medium containing calcium chloride added at 0, 50, or 100millimolar. One-half milliliter aliquots of the suspensions weredispensed into a 24-well microtiter plate.

Viability was judged by visualization under a light microscope. At 19hours, protoplasts of all five maize lines grown under the differentconditions appeared healthy, with large proportions of round, greencells; slightly more debris was observed in the 0 calcium conditions. At34 hours, protoplasts of all five maize lines showed a response to theincreased calcium conditions similar to what had been previouslyobserved; across the five maize lines, viability of protoplasts grownwithout added calcium was about 40%, while those grown with 50millimolar calcium was about 55%, and those grown with 100 millimolarcalcium was about 70%. These results demonstrate that culture conditionsincluding calcium cations at 50 or 100 millimolar improved viability ofprotoplasts from various maize germplasm over a culture time of 34hours.

Example 12

This example illustrates culture conditions effective in improvingviability and cell division rates of plant cells or plant protoplasts.More specifically, this non-limiting example describes the effects onmaize protoplast cultures of unconventionally hypoxic conditions. Thus,another aspect of the disclosure provides a method of improving the celldivision rate of a plant protoplast culture, wherein the cultureconditions include hypoxic conditions, for example, about one-halfnormal atmospheric oxygen concentrations; in certain embodiments of themethod, the culture conditions further include at least 40 millimolarCa²⁺ or Mg²⁺ in the medium. In certain embodiments, the cell divisionrate of the plant cells or plant protoplasts is improved by at least20%, or by at least 50%, or by at least 75%, or by at least 100%. In anembodiment, the culture conditions include at least 40 millimolar Ca′and about one-half normal atmospheric oxygen concentrations, and thecell division rate of the plant cells or plant protoplasts is improvedby at least 100% (i. e., cell division rate is about twice that observedin similar cultures grown under or subjected to normal atmosphericoxygen concentrations).

Normal atmospheric oxygen conditions are about 20.95% oxygen by volume;embodiments of the method thus include maintaining a plant cell or plantprotoplast culture at less than about 20% oxygen by volume, for example,at less than about 4%, less than about 6%, less than about 8%, less thanabout 10%, less than about 12%, less than about 14%, less than about16%, or less than about 18% oxygen by volume. In certain embodiments,the method involves maintaining a plant cell or plant protoplast culturebetween or about 5 to about 10% oxygen by volume, or even below about 5%oxygen by volume (e. g., at about 2%, about 3%, or about 4% oxygen byvolume).

Plant cell or protoplast cultures are conveniently maintained underhypoxia (oxygen concentration less than normal atmospheric oxygenconcentration) with incubation systems that use nitrogen gas pressure toincrease the percentage of nitrogen in the atmosphere with concomitantdecrease of the percentage of oxygen in the atmosphere. An oxygen sensoris used to control and rapidly re-equilibrate oxygen levels as needed.Commercial bioreactor or incubation systems (e. g., the “Avatar”bioreactor/incubator, XCell Biosciences, San Francisco, Calif.) can beemployed.

Initial experiments indicated that decreasing atmospheric oxygenconcentrations were beneficial to the plant cells or protoplasts.Similar effects of hypoxia were observed for cell cultures from severalmaize lines (HiIIA, B104, B73, A188, and BMS), as well as from kale.Cells incubated under normal ambient (about 21% by volume) oxygenconcentrations showed a brown-coloured phenotype (caused by productionof phenolic compounds, an indicator of cellular stress) faster thanthose incubated under 5% oxygen by volume. Cell division was monitored,e. g., by microscopic observations. For example, a greater number ofliving cells, resulting from cell division, were observed in culturesincubated under 5% or 10% oxygen by volume, when compared to culturesgrown under or subjected to normal atmospheric (about 21%) oxygen. Twoexperiments demonstrated that approximately twice as many living cellsresulting from cell division in cultures incubated under 5% oxygen byvolume, when compared to cultures grown under or subjected to about 21%oxygen by volume. Evidence of cell division was observed by microscopyand included increased cell or protoplast size, bulging membranes, andlarge groups of organelles occupying the separating daughter cells.Quantification of cell division was carried out using fluorescentstaining. Cells were treated with the thymidine analogue5-ethynyl-2′-deoxyuridine (EdU), which is incorporated into DNA duringS-phase and can be fluorescently activated using a fluorochrome that hasbeen conjugated to an azide molecule. Cells that are the result ofcellular division in the presence of EdU were identified by theresulting green fluorescent signal, which can be quantified by variousmeans, e. g., with a microplate reader, cell counter, or cell sorter.

In one experiment, maize A188 protoplasts were grown under either 5% or21% oxygen by volume in YPIM B-liquid medium containing 100 millimolarCa²⁺. On day 8 of culture, the culture grown under 5% oxygen by volumewere observed to have many more protoplasts displaying signs of celldivision (“budding” and bulging of membranes, with organellesdistributed among both forming daughter cells), in comparison to theculture grown under 21% oxygen by volume.

In a separate experiment, maize HiIIA protoplasts were grown in 24-wellplates using YPIM B-liquid medium containing 100 millimolar Ca²⁺ and 20micromolar EdU. Identical plates were incubated under hypoxia (5% oxygenby volume, 26 degrees Celsius, 80% relative humidity) or ambient oxygen(21% oxygen by volume, 26 degrees Celsius, 80% relative humidity).Entire wells (250 microliters) were taken for EdU detection at four timepoints. The contents of each well was centrifuged and the pelletsubjected to the Click-iT™ EdU Alexa Fluor™ 488 Imaging Kit (cataloguenumber C10337, Thermo Fisher Scientific, Waltham, Mass.) fluorescentlabelling protocol. The labelled protoplasts were resuspended in YPIMB-medium and samples analyzed on a Nexcelom Cellometer (NexcelomBioscience LLC, Lawrence, Mass.). This assay was developed to detect theEdU signal from the nucleus of a cell resulting from cell division, aswell as the fluorescent signal from chloroplasts as a measure of totalcell count. Results are provided in Table 4 as a percentage of cellsdisplaying the EdU signal relative to the total cell count (“% EdU”).The data indicate that the rate of cell division (expressed as % EdU)was increased by between about 2- to about 4-fold (about 100 to about400 percent increase) in the cultures grown under hypoxic conditions (5%oxygen by volume), compared to the rate of cell division in culturesgrown under normal atmospheric oxygen (21% by volume).

TABLE 4 % oxygen culture time by volume (hours) % EdU  5  93 12  93 —*117 19 117 19 141 11 141 33 165 19 165 15 21  93 6.5  93 —* 117 6.9 1178.0 141 13 141 4.8 165 5.0 165 0.0 *only single samples taken

Another experiment was carried out using protocols similar to those usedin the immediately preceding experiment but using maize B104 protoplastsgrown 24-well plates using YPIM B-liquid medium containing 100millimolar Ca²⁺ and 20 micromolar EdU, with or without 0.5 millimolarglutathione added to the medium. Entire wells (1000 microliters) weretaken for EdU detection at three time points; analyses were of duplicatesamples of each of two replicate wells. Results are provided in Table 5and illustrated in FIG. 1 as a percentage of cells displaying the EdUsignal relative to the total cell count (“% EdU”). The data indicatethat, in the cultures grown with 100 millimolar Ca²⁺ but no addedglutathione, the rate of cell division (expressed as % EdU) wasincreased by about 2- to 2.5-fold (about 100 to about 250 percentincrease) under hypoxic conditions (5% oxygen by volume), compared tothe rate of cell division in cultures grown under normal atmosphericoxygen (21% by volume). In the cultures grown under hypoxic conditions,the addition of 0.5 millimolar glutathione resulted in little change inthe rate of cell division (expressed as % EdU), but in the culturesgrown under normal atmospheric oxygen, the addition of 0.5 millimolarglutathione resulted in an increase in the rate of cell division(expressed as % EdU) of about 2-fold (about 100% increase), indicatingthat, under normal atmospheric conditions where greater oxidative stressis possible, the beneficial antioxidant effects of glutathione aresignificant.

TABLE 5 EdU staining O₂ (% Glutathione culture standard volume) Ca²⁺(mM) (mM) time (h) mean % deviation  5 100 0    90 21.9 5.2 138 27.8 1.0186 19.7 2.3 21 100 0    90 8.0 1.3 138 12.9 1.0 186 8.2 1.2  5 100 0.5 90 18.4 0.7 138 28.0 4.7 186 21.1 0.8 21 100 0.5  90 16.9 4.9 138 21.94.9 186 18.3* —* *mean of duplicate samples from single well

Example 13

This example illustrates methods and compositions effective in improvingthe efficacy of homology-directed repair (HDR) genome editing in plantcells or plant protoplasts. More specifically, this non-limiting exampledescribes improving the efficacy of homology-directed repair (HDR)genome editing in plant protoplasts by subjecting the protoplasts tounconventionally hypoxic conditions or by treating with a reactiveoxygen species (ROS) concentration-lowering agent. Thus, an aspect ofthe invention provides a method of improving the efficacy of HDR genomeediting in plant cells or plant protoplasts, by providing genome editingmolecules to a plant cell previously, concurrently, or subsequentlyexposed to a hypoxic condition, or to a ROS concentration-loweringagent, or to a combination thereof; in embodiments, the cultureconditions further include at least 40 millimolar Ca²⁺ or Mg²⁺ in themedium. Another aspect of the invention provides compositions comprisingat least one HDR genome editing agent (such as a sequence-specificnuclease, guide RNA, or donor template polynucleotide) and a plant cellor protoplast in which the ROS concentration is decreased (for example,by subjecting the protoplasts to unconventionally hypoxic conditions orby treating with a ROS concentration-lowering agent), relative to acontrol plant cell or protoplast; in embodiments, the compositionfurther includes a culture medium containing at least 40 millimolar Ca²⁺or Mg²⁺.

In this example, hypoxia is demonstrated to increase the efficiency ofHDR editing in a non-endogenous reporter gene. A “traffic light”reporter was designed to contain a blue fluorescent protein (BFP) and ared fluorescent protein “mCherry” in the construct encoding thepolyprotein BFP-LP4/2A-mCherry-NLS. LP4/2A is a hybrid linker peptidethat contains the first nine amino acids of LP4 and 20 amino acids of 2A(see doi[dot]org/10[dot]1371/journal[dot]pone[dot]0174804), and has highcleavage splicing efficiency within the polyprotein construct. To createthe “traffic light” reporter, two nucleotides are added in front ofLP4/2A to make the translation of mCherry out of frame. The DNA sequenceencoding the polyprotein BFP-LP4/2A-mCherry-NLS is provided as:

(SEQ ID NO: 10) ATGGTCAGCAAGGGAGAGGAGCTTTTCACGGGGGTGGTCCCCATCCTCGTGGAATTGGACGGCGATGTTAATGGGCACAAATTTTCCGTTTCTGGAGAGGGTGAGGGCGATGCGACATATGGGAAGTTGACCCTTAAATTTATCTGCACGACTGGCAAGCTCCCTGTCCCCTGGCCTACA CTTGTCACGACGTTGA CTCACGGAGTCCAGTGCTTTTCGAGGTATCCTGATCATATGAAACAGCACGATTTTTTCAAGTCAGCTATGCCCGAGGGGTATGTTCAGGAAAGAACTATCTTCTTTAAAGATGATGGCAATTACAAGACGAGAGCGGAGGTGAAGTTTGAGGGGGATACACTTGTTAATAGAATCGAACTGAAGGGAATCGACTTTAAGGAGGACGGAAACATACTGGGTCACAAACTTGAGTATAACTACAACTCTCACAATGTCTACATAATGGCGGACAAGCAGAAGAACGGTATTAAAGTCAACTTCAAAATCCGCCACAACATTGAGGACGGATCCGTCCAATTGGCCGATCATTACCAGCAAAATACTCCGATAGGTGACGGGCCCGTTTTGCTGCCCGATAATCACTATTTGTCCACCCAGTCCAAGCTCTCTAAGGATCCGAATGAGAAGAGAGACCATATGGTCCTCCTTGAGTTTGTTACCGCTGCGGGTATAACGCTTGGCATGGATGAACTTTACAAGTGtccaacgcggcggacgaggtggctacccagctgttgaattttgaccttcttaagcttgcgggagacgtcgagtccaaccctgggcctATGGTCAGCAAGGGCGAGGAGGACAATATGGCTATCATCAAGGAGTTCATGAGGTTTAAGGTTCACATGGAAGGCTCAGTCAACGGGCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCAGGCCTTACGAGGGCACCCAGACCGCTAAGCTGAAGGTGACGAAGGGCGGCCCCCTCCCTTTCGCCTGGGACATCCTGTCCCCGCAGTTCATGTACGGCAGCAAGGCCTACGTCAAGCACCCGGCGGACATCCCGGACTACCTCAAGCTGTCCTTCCCGGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTCACGGTCACCCAGGACTCCAGCCTCCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGGGGCACCAACTTCCCTTCGGACGGCCCGGTCATGCAGAAGAAGACGATGGGCTGGGAGGCTTCCTCGGAGAGGATGTACCCTGAGGACGGAGCCCTGAAGGGCGAGATCAAGCAGAGGCTCAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTGAAGACGACGTACAAGGCGAAGAAGCCTGTGCAGCTCCCGGGCGCATACAACGTCAACATCAAGCTGGACATCACGTCCCACAACGAGGACTACACGATCGTGGAGCAGTACGAGCGGGCGGAGGGGCGGCATAGCACGGGCGGGATGGACGAGCTGTACAAG cctaagaagaagaggaaggttTGA,where the BFP sequence is in bold, uppercase with the gRNA targetingregion is underlined; the LP4/2A sequence is in lowercase, the mCherrysequence is in italicized uppercase; and the nuclear localization signal(NLS) is double underlined and in lowercase. An HDER donor template withthe DNA sequenceAAGTTGACCCTTAAATTTATCTGCACGACTGGCAAGCTCCCTGTCCCCTGGCCTACACTTGTCACGACGTTGACTTACGGAGTCCAGTGCTTTTCGAGGTATCCTGATCATATGAAACAGCACGATTTTTTCAAGTCAGCTATG (SEQ ID NO:11, Integrated DNA Technologies, Coralville,Iowa) was provided as a single-stranded DNA (ssDNA), phosphorylated onthe 5′ end and containing three phosphorothioate linkages at eachterminus (i. e., the three linkages between the most distal four baseson either end of the strand). A guide crRNA with the sequenceCUUGUCACGACGUUGACUCAGUUUUAGAGCUAUGCU (SEQ ID NO:12, BFP guide crRNA;Integrated DNA Technologies, Coralville, Iowa) was designed to introducea double-stranded break (DSB) within the “traffic light” reporter's BFPcoding sequence. This DSB can be repaired through the NHEJ pathway withan indel that leads to in-frame expression of mCherry, resulting in redfluorescence. Alternatively, if the donor template is provided andintegrated at the DSB by the HDR pathway, this results in a change ofHis67 to Tyr in the original BFP sequence, changing the BFP reporter toa green fluorescent protein (GFP).

To prepare a guide RNA duplex, 63 microliters of 100 micromolar the BFPguide crRNA (SEQ ID NO:12) was added to 63 microliters of 100 micromolartracrRNA (Integrated DNA Technologies, Coralville, Iowa), heated to 95degrees Celsius for 5 minutes, and then removed from the heating blockand allowed to cool to room temperature. Before transfection, 18microliters (180 milligrams) of Cas9 protein (Aldevron, Fargo, N. Dak.)was added to the gRNA complex and the mixture allowed to incubate for 5minutes at room temperature to form the ribonucleoprotein (RNP) complex;4.5 microliters (45 micrograms) of salmon sperm DNA was added to the RNPsolution.

Transfections were carried out as follows. Maize B73 plant protoplastcells were prepared essentially as described in Example 1. Theprotoplasts were at a concentration of 2×10{circumflex over ( )}5protoplasts/milliliter. About 40×10{circumflex over ( )}4 protoplasts in2004 of MMg solution were used in each transfection experiment. To eachtube was added 14 microliters of the RNP solution, 10 microliters of thereporter plasmid, with or without 10 microliters of the donor template,plus 244 microliters of 40% PEG and sufficient buffer if needed toequalize volumes; the tubes were tapped to mix and allowed to incubatefor 5 minutes at room temperature. The reaction was stopped by additionof 976 microliters of maize washing buffer. The protoplast cells werecentrifuged at 1200 rpm for 2 minutes, and the supernatant was removed.The pelleted cells were then resuspended in PIM medium containing 50millimolar CaCl₂ and then plated in 6-well plates coated with 5% calfserum. The plates were sealed with Parafilm™ and allowed to incubate at37 degrees Celsius for 1 hour. For the hypoxia treatment, the Parafilm™was removed and the plates were then placed in a hypoxia chamber havingabout 5% oxygen by volume at 26 degrees Celsius in the dark. Fornormoxia treatment, the Parafilm™ was kept in place and the plates wereincubated at 26 degrees Celsius in a growth chamber in the dark. Cellswere harvested 48 hours after transfection for imaging on a fluorescentmicroscope, which allows quantification of HDR editing efficiency bymeasuring GFP fluorescence based either on individual cell fluorescenceor on averaged fluorescence (“relative fluorescence units”, RFU)measured across a well.

In a first experiment using these procedures, HDR editing frequency wasquantitated by individual cell fluorescence. The HDR editing frequencyunder normoxic conditions was 1.97% of the cell population. HDR editingfrequency was increased by hypoxia treatment to 3.91% of the cellpopulation (about a 2-fold increase over normoxia). In a secondexperiment using these procedures, HDR editing frequency was quantitatedby averaged fluorescence measured across a well and expressed as an RFUratio normalized to the RNP-only (no donor template) control. No markeddifference was observed between the NHEJ editing frequency undernormoxic conditions (1.11) and under hypoxic conditions (1.02), but theHDR editing frequency observed under normoxic conditions (0.897)increased about 9-fold under hypoxic conditions (8.19). The data fromthese experiments indicate that exposure of the plant cells to hypoxia,which is expected to reduce reactive oxygen species (ROS) concentrationsin the cells, increased efficacy of homology-directed repair (HDR) of atarget gene in the plant cells.

Example 14

This example illustrates methods and compositions effective in improvingthe efficacy of homology-directed repair (HDR) genome editing in plantcells or plant protoplasts. More specifically, this non-limiting exampledescribes improving the efficacy of homology-directed repair (HDR)genome editing in plant protoplasts by subjecting the protoplasts tounconventionally hypoxic conditions or by treating with a reactiveoxygen species (ROS) concentration-lowering agent. Thus, an aspect ofthe invention provides a method of improving the efficacy of HDR genomeediting in plant cells or plant protoplasts, by providing genome editingmolecules to a plant cell previously, concurrently, or subsequentlyexposed to a hypoxic condition, or to a ROS concentration-loweringagent, or to a combination thereof; in embodiments, the cultureconditions further include at least 40 millimolar Ca²⁺ or Mg²⁺ in themedium. Another aspect of the invention provides compositions comprisingat least one HDR genome editing agent (such as a sequence-specificnuclease, guide RNA, or donor template polynucleotide) and a plant cellor protoplast in which the ROS concentration is decreased (for example,by subjecting the protoplasts to unconventionally hypoxic conditions orby treating with a ROS concentration-lowering agent), relative to acontrol plant cell or protoplast; in embodiments, the compositionfurther includes a culture medium containing at least 40 millimolar Ca²⁺or Mg²⁺.

In this example, hypoxia is demonstrated to increase the efficiency ofHDR editing in an endogenous plant gene, the maize (Zea mays) alcoholdehydrogenase ADH1 gene with the partial genomic sequence provided as:

(SEQ ID NO: 13) GAACAGTGCCGCAGTGGCGCTGATCTTGTATGCTATCCTGCAATCGTGGTGAACTTATTTCTTTTATATCCTTTACTCCCATGAAAAGGCTAGTAATCTTTCTCGATGTAACATCGTCCAGCACTGCTATTACCGTGTGGTCCATCCGACAGTCTGGCTGAACACATCATACGATCTATGGAGCAAAAATCTATCTTCCCTGTTCTTTAATGAAGGACGTCATTTTCATTAGTATGATCTAGGAATGTTGCAACTTGCAAGGAGGCGTTTCTTTCTTTGAATTTAACTAACTCGTTGAGTGGCCCTGTTTCTCGGACGTAAGGCCTTTGCTGCTCCACACATGTCCATTCGAATTTTACCGTGTTTAGCAAGGGCGAAAAGTTTGCATCTTG ATGATTTAGCTTGACTATGCGATTGCTTTCCTGGACCCGTGCAGCTGCGGTGGCATGGGAGGCCGGCAAGCCACTGTCGATCGAGGAGGTGGAGGTAGCGCCTCCGCAGGCCATGGAGGTGCGCGTCAAGATCCTCTTCACCTCGCTCTGCCACACCGACGTCTACTTCTGGGAGGCCA AGGTATCTAATCAGCCATCCCATTTGTGATCTTTGTCAGTAGATATGATACAACAACTCGCGGTTGACTTGCGCCTTCTTGGCGGCTTATCTGTCTTAGGGGCAGACTCCCGTGTTCCCTCGGATCTTTGGCCACGAGGCTGGAGGGTA.The first exon located at nucleotide positions 409-571 is indicated bybold, underlined text.

A ribonucleoprotein (RNP) was prepared with Cas9 nuclease (Aldevron,Fargo, N. Dak.) and a guide RNA complex designed to edit the first ADH1exon, consisting of a crRNA (ZmADH1-B) with the sequenceGGCCUCCCAGAAGUAGACGUGUUUUAGAGCUAUGCU (SEQ ID NO:14) complexed with atracrRNA (both purchased from Integrated DNA Technologies, Coralville,Iowa). A single-stranded DNA donor template designed forhomology-directed repair of the ADH exon had the sequenceAGGAGGTGGAGGTAGCGCCTCCGCAGGCCATGGAGGTGCGCGTCAAGATCCTCTTCACCTCGCTCTGGTACCCCGACGTCTACTTCTGGGAGGCCAAGGTATCTAATCAGCCATCCCATTTGTGATCTTTGTCAGTAGATATGA (SEQ ID NO:15, Integrated DNA Technologies, Coralville,Iowa); the donor ssDNA contained three phosphorothioate linkages at eachterminus (i. e., the three linkages between the most distal four baseson either end of the strand), and contained a KpnI restriction enzymedigestion site at nucleotide positions 67-72 (shown as underlined text).A double-stranded DNA (purchased from Integrated DNA Technologies,Coralville, Iowa) was designed with a forward strand having the sequenceGTTTAATTGAGTTGTCATATGTTAATAACGGTAT (SEQ ID NO:16) and a reverse strandhaving the sequence ATACCGTTATTAACATATGACAACTCAATTAAAC (SEQ ID NO:17),wherein each strand was phosphorylated on the 5′ end and contained twophosphorothioate linkages at each terminus (i. e., the two linkagesbetween the most distal three bases on either end of the strand); thiscontained an NdeI restriction site located at nucleotide positions 16-21of the forward strand (SEQ ID NO:16), shown as underlined font.Insertion of this sequence into the first exon of the target gene ADH1serves as a proxy readout for efficiency of the NHEJ pathway (i.e.,non-homologous repair of the expected DSB).

Transfection procedures were essentially as described in Example 13. Ina first experiment, cells were treated with the HDR ssDNA donorcontaining a KpnI restriction site, or with no donor polynucleotide.After transfection with the editing reagents, the washed and pelletedcells were then resuspended in PIM containing 50 millimolar CaCl₂, andthen plated in 6-well plates coated with 5% calf serum. The plates weresealed with Parafilm™ and allowed to incubate at 37 degrees Celsius for1 hour. For the hypoxia treatment, the Parafilm™ was removed and theplates were then placed in a hypoxia chamber having about 5% oxygen byvolume at 26 degrees Celsius in the dark. For normoxia treatment, theParafilm™ was kept in place and the plates were incubated at 26 degreesCelsius in a growth chamber in the dark. Cells were harvested 48 hoursand HDR editing efficacy was quantitated by PCR analysis using Phusion™Flash PCR (Thermo Fisher Scientific, Waltham, Mass.). Under normoxicconditions, HDR editing efficacy was 0%. Under hypoxic conditions, HDRediting efficacy was increased to 1.35%. Thus, treatment with hypoxia,which is expected to reduce reactive oxygen species (ROS) concentrationsin the cell, increased HDR editing efficiency in an endogenous plantgene.

In a second experiment, cells were treated with the HDR ssDNA donorcontaining a Kpnl restriction site, or with the NHEJ dsDNA containing anNdeI restriction site, or with no donor polynucleotide. Aftertransfection with the editing reagents, the washed and pelleted cellswere then resuspended in PIM containing 50 millimolar CaCl₂, with orwithout 0.5 millimolar glutathione (GSH), and then plated in 6-wellplates coated with 5% calf serum. The plates were sealed with Parafilm™and allowed to incubate at 37 degrees Celsius for 1 hour. For thehypoxia treatment, the Parafilm™ was removed and the plates were thenplaced in a hypoxia chamber having about 5% oxygen by volume at 26degrees Celsius in the dark. For normoxia treatment, the Parafilm™ waskept in place and the plates were incubated at 26 degrees Celsius in agrowth chamber in the dark. Cells were harvested 48 hours and subjectedto PCR analysis using Phusion™ Flash PCR (Thermo Fisher Scientific,Waltham, Mass.). Results are provided in Table 6. Under normoxicconditions, no HDR editing was detected in cells treated with thehomologous HDR donor, in contrast to cells treated with thenon-homologous NHEJ donor, where 24% NHEJ editing efficiency wasobserved. With treatment with glutathione, HDR efficiency was 5.0% andNHEJ efficiency was about 15%. Under hypoxia, HDR efficiency was 3.25%and NHEJ efficiency was about 14%. These data indicate that treatmentwith a low-molecular-weight thiol antioxidant or with hypoxia,treatments that are expected to reduce reactive oxygen species (ROS)concentrations in the cell, increased HDR editing efficiency in anendogenous plant gene.

TABLE 6 % KpnI % NdeI Treatment Editing Reagents (HDR) (NHEJ) NormoxiaNull 0.00  0.00 Normoxia RNP only 0.00  0.00 Normoxia RNP plus HDR donor0.00 — Normoxia RNP plus NHEJ donor — 24.00 0.5 mM GSH Null 0.00  0.000.5 mM GSH RNP only 0.00  0.00 0.5 mM GSH RNP plus HDR donor 5.00 — 0.5mM GSH RNP plus NHEJ donor — 15.01 Hypoxia Null 0.00  0.00 Hypoxia RNPonly 0.00  0.00 Hypoxia RNP plus HDR donor 3.25 — Hypoxia RNP plus NHEJdonor — 14.35

All cited patents and patent publications referred to in thisapplication are incorporated herein by reference in their entirety. Allof the materials and methods disclosed and claimed herein can be madeand used without undue experimentation as instructed by the abovedisclosure and illustrated by the examples. Although the materials andmethods of this disclosure have been described in terms of embodimentsand illustrative examples, it will be apparent to those of skill in theart that substitutions and variations can be applied to the materialsand methods described herein without departing from the concept, spirit,and scope of the disclosure. For instance, while the particular examplesprovided illustrate the methods and embodiments described herein using aspecific plant, the principles in these examples are applicable to anyplant of interest. All such similar substitutes and modificationsapparent to those skilled in the art are deemed to be within the spirit,scope, and concept of the disclosure as encompassed by the embodimentsrecited herein and the specification and appended claims.

1.-6. (canceled)
 7. A method for making a maize plant protoplast havinga genomic modification comprising: (a) providing genome editingmolecules to a maize plant protoplast lacking a cell wall; wherein thegenome editing molecules are designed for homology-directed repair of atarget gene in the plant cell's genome and comprise: (i) an RNA-guidednuclease and a guide RNA and a donor template polynucleotide; (ii) asequence-specific endonuclease and a donor template polynucleotide;(iii) one or more polynucleotides encoding an RNA-guided nuclease and aguide RNA and a donor template polynucleotide; (iv) a polynucleotideencoding a sequence-specific endonuclease and a donor templatepolynucleotide; or (v) any combination thereof; (b) exposing the maizeplant protoplast from step (a) to plant cell culture medium comprising areactive oxygen species (ROS) concentration lowering agent at aconcentration of 0.1 millimolar to 10 millimolar and Ca2+ and/or Mg2+ ata concentration of about 40 mM to 150 mM for at least one hour to effecthomology-directed repair of the target gene in the plant cell's genomeat a frequency that is increased by at least 2-fold in comparison to acontrol method wherein a control plant cell is provided with the genomeediting molecules but is not subjected to a ROS concentration loweringagent at a concentration of 0.1 millimolar to 10 millimolar; and, (c)isolating, selecting, identifying, and/or propagating a maize plant cellprotoplast comprising the genome modification, thereby making the maizeplant protoplast cell having a genomic modification. 8.-10. (canceled)11. The method of claim 7, wherein the ROS concentration lowering agentis one or more low-molecular-weight thiol compound(s) wherein themolecular weight of the thiol compound is less than 1,000.
 12. Themethod of claim 11, wherein the thiol compound(s) are glutathione,cysteine, cysteinyl glycine, gamma-glutamyl cysteine, N-acetylcysteine,cysteine, thiocysteine, homocysteine, lipoic acid, dithiothreitol, or acombination thereof.
 13. The method of claim 11, wherein the thiolcompound is gluthathione. 14.-20. (canceled)